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Restorative Neurology and Neuroscience 33 (2015) 95–104 DOI 10.3233/RNN-140430 IOS Press
Neuroprotective effects of collagen matrix in rats after traumatic brain injury Samuel S. Shina,b,c , Ramesh Grandhib , Jeremy Henchira,b , Hong Q. Yana,b,c , Stephen F. Badylakd and C. Edward Dixona,b,c,e,∗ a Brain
Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania of Neurological Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania c Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, Pennsylvania d McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania e Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania b Department
Abstract. Purpose: In previous studies, collagen based matrices have been implanted into the site of lesion in different models of brain injury. We hypothesized that semisynthetic collagen matrix can have neuroprotective function in the setting of traumatic brain injury. Methods: Rats were subjected to sham injury or controlled cortical impact. They either received extracellular matrix graft (DuraGen) over the injury site or did not receive any graft and underwent beam balance/beam walking test at post injury days 1–5 and Morris water maze at post injury days 14–18. Animals were sacrificed at day 18 for tissue analysis. Results: Collagen matrix implantation in injured rats did not affect motor function (beam balance test: p = 0.627, beam walking test: p = 0.921). However, injured group with collagen matrix had significantly better spatial memory acquisition (p < 0.05). There was a significant reduction in lesion volume, as well as neuronal loss in CA1 (p < 0.001) and CA3 (p < 0.05) regions of the hippocampus in injured group with collagen matrix (p < 0.05). Conclusions: Collagen matrix reduces contusional lesion volume, neuronal loss, and cognitive deficit after traumatic brain injury. Further studies are needed to demonstrate the mechanisms of neuroprotection by collagen matrix. Keywords: Collagen, matrix, traumatic brain injury, bioscaffold, neuroregeneration, neurorepair
1. Introduction Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Long-term neurobehavioral deficits are common among survivors of TBI, but only a few pharmacological agents such as Amantadine have shown success in improving neurobehavioral function after TBI (Giacino et al., 2012). Strategies to increase neuroregeneration (Brazda and Muller, 2009) ∗ Corresponding
author: C. Edward Dixon, PhD, Department of Neurological Surgery, University of Pittsburgh, 201 Hill Building, 3434 Fifth Avenue, Pittsburgh, PA 15260, USA. Tel.: +1 412 383 2188; Fax: +1 412 624 0943; E-mail: [email protected].
and reduce inflammation (Kumar and Loane, 2012) after traumatic injury to the central nervous system (CNS) have been previously reviewed. However, clinical treatment options for TBI are limited due to the heterogeneity of the disease and the irreversibility of injury to neural structures. DuraGen (DuraGen Plus, DP-1011) is a semisynthetic Type I collagen matrix from bovine deep flexor tendon with delicate mechanical properties. Although collagen matrices such as DuraGen have been commonly used for dural repair, several recent studies have explored the use of collagen matrices and biological scaffold ECMs in repair of neural injury. Collagen
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matrices have been used as a platform for therapeutic delivery of stem cells in experimental models of ischemia (Matsuse et al., 2011; Park et al., 2002), spinal cord injury (Bakshi et al., 2004), and TBI (Lu et al., 2007; Tate et al., 2002; Xiong et al., 2009). These matrices have been shown to provide an effective foundation by which stem cells can anchor after tissue injury, leading to improved cell survival, neovascularization, and functional recovery. Biodegradable materials releasing growth factors have been utilized in the past to improve nerve regeneration (Nordblom et al., 2012). Even without seeding of neuronal cells or addition of factors such as brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), or fibronectin (FN), implantation of collagen composite hydrogels into adult rat brains had penetration by neural tissue (Woerly et al., 1990). Urinary bladder matrix (UBM) derived from porcine bladder also contains collagen, fibronectin, and various growth factors. In a recent study, injection of UBM into the injury site after TBI reduced both lesion volume and white matter injury (Zhang et al., 2013). In addition, there was an improvement in vestibulomotor function. In fact, recent study has shown that even implantation of collagen-based scaffolds alone can lead to increased levels of GDNF and BDNF, as well as proliferation and migration of neural precursor cells after brain injury (Huang et al., 2012). The current study aimed to determine if there is a beneficial effect of a readily available, FDA-approved collagen matrix graft in the injury site after TBI in rats, and the results demonstrated that collagen matrix may reduce the degree of injury and induce functional deficits.
2. Materials and methods 2.1. Ethics statement All animal work in this study has been conducted according to ethical national/international guidelines. The experiments in this study have been approved by Institutional Animal Care and Use Committee of the University of Pittsburgh.
light:dark cycle and given food and water ad libitum. Rats were divided into 4 groups: TBI rats with collagen matrix graft (IC), sham rats with collagen matrix graft (SC), TBI rats with no graft (IN), and sham rats with no graft (SN). For tissue staining studies, 39 animals were used: IC = 10, IN = 9, SN = 9, and SC = 11. For behavioral experiments, 48 animals were used: IC = 13, IN = 11, SN = 13, and SC = 11. 2.3. Surgical procedures for controlled cortical impact (CCI) Animals were injured by Controlled Cortical Impact (CCI) as previously described (Dixon et al., 1991). Briefly, rats were anesthetized with 5% isoflurane (IsoFlo; Abbott Laboratories, North Chicago, IL), carried in a 2:1 ratio of N2 O/O2 , endotracheally intubated, and maintained on a small-animal ventilator (Harvard Rodent Ventilator, Model 683; Harvard Apparatus, Holliston, MA) with 2% isoflurane in the same carrier gas mixture. Body temperature was maintained at 37◦ C. After placement in a stereotactic frame (David Kopf Instruments, Tujunga, CA), a parasagittal craniectomy 8 mm in diameter was performed, and TBI was induced with a 6-mm in diameter beveled impactor tip (impact speed: 4 m/s, injury depth: 2.6 mm, dwell time: 50 msec). The CCI device produced cortical contusion 8 mm in diameter. For IC and SC animals, a piece of collagen matrix (DuraGen, Integra LifeSciences Corporation, Plainsboro, NJ) about 8 mm in diameter was placed over the lesion site just after injury and entirely covering the craniotomy site. CCI at this injury severity, tears the dura, thus allowing direct contact of the collagen matrix with injured cortical tissue. The collagen matrix used in this study did not contain any chemo-attractants or growth factors, The exposed area was then closed by suturing the scalp while holding the 0.3 mm thick piece of matrix firmly against the injury site. For IN and SN animals, no graft material was used at the lesion site. Rats were allowed to recover from anesthesia before returning to their housing facility. In sham rats (SN, SC), craniectomy without CCI was performed. 2.4. Behavioral experiments: Motor test
2.2. Animals Eighty-seven male Sprague-Dawley rats (Harlan Laboratories Inc., Indianapolis, IN, USA) weighing 280–300 g were used. Rats were housed in 12:12 hour
On days 1–5 post injury, motor functions were tested using beam balance and beam walking tests, as previously described using our standard behavioral protocol (Singleton et al., 2010). Each of the four groups
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consisted of n = 10–13 animals. For beam balance test, rats were placed on a suspended wooden beam (width: 1.5 cm), and their latency to stay on the beam was recorded up to maximum of 60 seconds. Rats were assessed prior to injury, and then tested for 5 consecutive days after injury. For beam walking test, the latency of travel across a narrow wooden beam (length: 100 cm, width: 2.5 cm) to move away from loud white noise and bright environment into a darkened goal box at the end of the beam was measured. There are four metal pegs (diameter: 3 mm, height: 4 cm) to challenge the coordination and agility of rats during the travel. Two days before injury, rats have been trained to traverse the beam. For 5 days after injury, latency of travel was recorded up to maximum of 60 seconds. All motor and cognitive testing was performed blinded to experimental group designation. 2.5. Behavioral experiments: Spatial memory test A variant of the Morris water maze (MWM) (Morris et al., 1984) was employed as described previously on days 14–18 after injury (Dixon et al., 1994, 1999; Hamm et al., 1992; Scheff et al., 1997) in the same rats that had undergone motor testing. The water maze used a 120 cm-diameter and 60cmhigh plastic pool filled with 25◦ C water to a depth of 28 cm. A platform 10 cm in diameter and 27 cm high, 1 cm below the water’s surface, was used as the hidden goal platform. The pool was located in a 2.5 × 2.5 m room with numerous extramaze cues that remained constant during testing. A video tracking system (AnyMaze, Stoelting, Inc. Wood Dale, IL) recorded and quantitated the swimming motions of the rats. The hidden platform version of the MWM was used to assess spatial memory acquisition. Fiveday acquisition blocks consisted of four daily trials over 5 consecutive days. Each of the four groups consisted of n = 10–13 animals. Rats started a trial from each of the four possible start locations in random order. The location of the platform was held constant for each animal and was varied between animals. Rats were given a maximum of 120 seconds to find the hidden platform. All rats were allowed to remain on the platform for 30 seconds before being placed in a heated incubator between trials (4-minute inter-trial interval). A single probe trial to determine memory retention was performed on day 5 of MWM testing, in which the platform was removed and the percentage time in the platform quadrant and swim
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speed was measured for 60 sec by the video tracking system. 2.6. Tissue preparation, lesion volume analysis, hippocampal cell counting After completion of behavioral tests, rats were sacrificed with an overdose of sodium pentobarbital (100 mg/kg, intraperitoneal) before delivering 0.9% saline then 10% phosphate-buffered formalin intracardially. Brain tissue was dissected and post fixed in 10% phosphate buffered formalin for 5 days. The forebrain was paraffin-embedded and sectioned using a microtome (Leitz 1512; Leica Microsystems, Germany) at 7 m thickness every 500 m. The sections were then de-paraffinized, hydrated, and stained with hematoxylin and eosin (H & E) and coverslipped for lesion volume analysis. Using a light microscope, coronal sections of rat brain tissue were analyzed with imaging software (Image J version 1.46r, National Institutes of Health, Bethesda, MD, USA). Contusion volume was outlined on each section and lesion volume was calculated using Cavalieri principle. Surviving neurons in CA1 and CA3 regions of hippocampi were counted as described previously (Exo et al., 2009; Shellington et al., 2011). The number of neurons was counted on H & E stained sections by a blinded observer using light microscopy. Glial fibrillary acidic protein (GFAP) and trichrome stains were performed for the (Figs. 5, 6). 2.7. Methods for GFAP immunofluorescence staining on ECM sections Whole frontal brains were embedded in paraffin and then sectioned at 5m. Sections were mounted on SuperFrost Plus microscope slides and allowed to dry for 24 hours or more. Deparaffinization of the sections was done with 3 washes of xylene for 5 minutes each. Sections were then hydrated with 2 washes of 100% EtOH and 2 washes of 95% EtOH for 3 minutes each. Slides then were washed for 3 minutes in 1× TBS Automation Wash Buffer (BioCare Medical #TWB945 M) and then 3 minutes in distilled water. Antigen retrieval was then performed using BioCare Medical #CB910 M Antigen Decloaker by 5 minutes of medium power heating in a microwave followed by a 20 minute cooling off period. Once the antigen decloaker has cooled, a 5 minute distilled water wash followed by a 5 minute Automation Buffer wash was performed. The slides were then incubated at room
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temperature for 30 minutes with 10% Normal (donkey, Sigma #D9663) Blocking Serum. Slides were then incubated at 4◦ C overnight with GFAP (Millipore #MAB360) diluted in 5% Normal Blocking Serum. On the following day, the slides were allowed to acclimate to room temperature for 15 minutes followed by 2 washes for 10 minutes each in Automation Buffer. Slides were then incubated for 1 hour at room temperature with the secondary antibody (Life Technologies #A-21203). After incubation of the secondary antibody, 3 washes of 5 minutes each were performed followed by coverslipping with Vectashield Hardset.
volumes of each group were compared using Student’s T test. Morris water maze probe trial data and hippocampal neuronal counts were analyzed using one-way analysis of variance (ANOVA). When a significant difference between the groups was found by one-way ANOVA, post hoc analysis was performed using Student-Newman-Keuls method. Motor testing and Morris water maze results were analyzed using repeated measures ANOVA and Student-NewmanKeuls post hoc analysis.
3. Results 2.8.. Methods for masson trichrome stain After whole frontal brains were embedded in paraffin then sectioned at 5m, sections were mounted on SuperFrost Plus microscope slides and allowed to dry for 24 hours or more. We used the NovaUltra Masson Trichrome Stain Kit from IHCWorld (#IW3006). Deparaffinization of the sections was done with 2 washes with xylene for 10 minutes each. Slides were hydrated with 2 washes of 100% EtOH for 5 minutes followed by washes in 95% EtOH and 70% EtOH for 1 minute each. Slides were then rinsed in distilled tap water, and then post-fixed in Bouin’s Solution for 1 hour in a 56◦ C oven. Slides then were rinsed with running tap water for 5–10 minutes, and then stained with Weigert’s Iron Hematoxylin Solution for 10 minutes. Following Weigert’s Hematoxylin, the slides were rinsed in running tap water for 5–10 minutes and then washed in distilled water. Slides were then stained with Biebrich Scarlet-Acid Fuchsin Solution for 10–15 minutes, and then washed in distilled water. Slides were then differentiated with PP Acid Solution for 10–15 minutes and then transferred directly into Aniline Blue Solution for 5–10 minutes. Slides were then rinsed briefly in distilled and differentiated in Acetic Acid Solution for 2–5 minutes. Following a wash in distilled water, the slides were dehydrated with 1 wash of 95% EtOH for 1 minute and then 2 washes of 100% EtOH for 1 minute each. Slides were then washed in xylene twice for 5 minutes each, and then coverslipped with Permount (Fisher Scientific #SP-15). 2.9. Statistical analysis Statistical testing was performed using SPSS software (v. 20, SPSS Inc., Chicago, IL, USA). Lesion
Repeated measures ANOVA showed significant differences in group (F3,47 = 21.796, p < 0.001) and day (F5,235 = 23.812, p < 0.001) comparisons on beam balance testing on days 1–5 (Fig. 1a), as well as group × day interaction (F15,235 = 6.721, p < 0.001). Post hoc analysis showed that although both injured groups were significantly different from both sham groups (p < 0.001), there was no significant difference between the IC and IN groups (p = 0.627). In beam walking test (Fig. 1b), there were significant differences in group (F3,47 = 78.404, p < 0.001), day (F3,47 = 70.900, p < 0.001), and group x day interaction (F15,235 = 9.479, p < 0.001). Similar to beam balance testing, both the injured groups were significantly different from both sham groups (p < 0.001), but there was no significant difference between IC and IN groups (p = 0.921). Spatial working memory was assessed using MWM at a later time point on days 14–18 (Fig. 2). Time course of latency to find the platform for 5 consecutive days was used to assess spatial learning. Repeated measures ANOVA showed group (F3,44 = 14.076, p < 0.001) and day (F4,176 = 27.798, p < 0.001) differences. However, there was no group x day interaction (F12,176 = 1.377, p = 0.181). Post hoc analysis revealed significant reduction in latency in IC group compared to IN group (p < 0.05) as well as significant differences between SN and SC groups compared to IN group (p < 0.01). Spatial memory retention was assessed by using probe trials, which showed significant group differences (F3,44 = 3.616, p = 0.02). By post hoc analysis, there was a significant difference between duration spent in target quadrant by IN (18.0 ± 1.6 seconds) and SN (25.5 ± 2.1 seconds) groups (p < 0.05), showing injury effect on spatial memory retention. However, there was no significant difference between IC and
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Fig. 1. Motor function. The motor data was assessed by beam balance test (A) and beam walking test (B). In both experiments, IC and IN were significantly different from SN (∗ p < 0.001).
Fig. 2. Spatial working memory assessed by Morris water maze test. There is a significant improvement in the latency of the injured rats after collagen matrix treatment (A) (∗ p < 0.05, ∗∗ p < 0.01 compared to IN). Probe trial showed decreased duration spent in target quadrant in injured animals (∗ p < 0.05), but no effect of collagen matrix on memory retention (B).
IN groups (p = 0.81), indicating no effect of collagen matrix on memory retention after injury. Also, there were no significant differences among groups for swim speed, indicating that the performance differences in water maze were not due to physical disability (F3,44 = 0.744, p = 0.53). The average swim speed were as follows: IC = 0.269 ± 0.008, IN = 0.269 ± 0.011, SN = 0.278 ± 0.011, SC = 0.287 ± 0.007. After sacrifice of the rats, tissues were analyzed using light microscopic morphometry. Lesion volume comparisons showed a significant reduction in injuryinduced lesion volume (p < 0.05) in IC rats compared to
IN rats (Fig. 3). The degree of injury was also assessed by counting the number of neurons in CA1 and CA3 regions of the hippocampus (Fig. 4). For CA1 neuronal counts, there was a significant between group difference (F3,35 = 28.611, p < 0.001). There was a significant reduction in both IN and IC groups’ neurons compared to SN (p < 0.001). Also, collagen matrix was effective in attenuating neuronal losses: IC compared to IN showed significant difference (p < 0.001). For CA3 neuronal counts, there was also a significant between group difference (F3,35 = 18.551, p < 0.001). The IC and IN groups showed significant reduction
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Fig. 3. Tissue lesion after injury. The histological samples were observed after no treatment (A), or collagen matrix treatment (B). Quantified lesion volume is also presented (C). There was significant reduction of lesion size in injured animals that received collagen matrix (∗ p < 0.05).
Fig. 4. Hippocampal neuronal counts. There was a significant decrease in the number of neurons in injured groups in both CA1 (A) and CA3 (B) regions. Collagen matrix treatment significantly attenuated this neuronal loss compared to untreated injured controls. (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).
in the number of surviving neurons compared to the SN group (p < 0.01 and p < 0.001, respectively). Collagen matrix also attenuated the neuronal loss in CA3: IC compared to IN showed significant difference (p < 0.05). Several other studies were performed to further understand the effect of collagen matrix. Frozen sections were stained for glial fibrillary acidic
protein (GFAP), showing no difference in IC and IN in terms of astrocytes in the pericontusional areas (Fig. 5). Also, in order to visualize whether collagen matrix was still present or not, trichrome staining was performed showing no evidence of remaining collagen matrix after sacrifice at 3 weeks (Fig. 6).
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Fig. 5. GFAP staining through the injured region shows no increase in the number of astrocytes among TBI rats treated with collagen matrix (Left) compared to TBI rats with no treatment (Right).
Fig. 6. Two sections through the hippocampus of injured rats treated with collagen matrix show no residual matrix overlying the injured region on trichrome stain.
4. Discussion In this study, we applied the collagen-based matrix to the area of cortical injury of rats after trauma. This treatment in injured rats resulted in improvement of spatial memory acquisition, and histological sections
revealed reduced lesion volume as well as increased hippocampal neuronal counts. These histological sections showed no remaining collagen matrix at the site of injury. Thus, its neuroprotective effect in TBI may be due to its activity as biological scaffold ECMs enhancing repair of neural injury rather than repairing the
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dural defect. In fact, the surgical procedure in this study is not a dural repair procedure, since it entailed implantation of collagen matrix at a width of an injury cavity. Collagen matrix implanted was only large enough to fit into the injury cavity following TBI, and did not cover overlap into the intact parts of the dura as a dural repair procedure would have. Future studies are needed to elucidate the mechanisms by which collagen matrix conferred benefit in the present study. Most studies involving the use of biological scaffold matrices in experimental brain injury models have employed a strategy in which stem cells have been implanted in the local environment with the addition of exogenous trophic factors. Impregnation of growth factors within the ECM or delivery of trophic factors by microspheres optimizes the microenvironment for neural growth and stimulates neurogenesis, proliferation, migration, and neural survival. Collagen matrices with impregnated nerve growth factor (NGF) and BDNF can enhance neurite outgrowth (Labour et al., 2012), and microspheres that release basic fibroblast growth factor (FGF2) can improve cell survival and behavioral recovery after cerebral ischemia (Matsuse et al., 2011). In addition, the implantation of a collagen matrix can increase BDNF and GDNF (Huang et al., 2012), leading to cell proliferation and functional recovery. The use of collagen as a scaffold, without the concomitant presence of trophic factors, has been shown to stabilize and optimize the survival and differentiation of neuronal stem cells after local transplantation (Woerly et al., 1990; Huang et al., 2012; Yu et al., 2010). Importantly, collagen matrix by itself may result in neural growth and functional recovery. Acellular and denaturized collagen matrices have been shown to promote neural growth (Zhang et al., 2013; Huang et al., 2012; Rabinowitz et al., 2005), showing that these matrices alone may be biologically active. An In vitro study of collagen matrix has demonstrated the ability of the synthetic dural matrix to support growth of axons and dendrites in neuronal cultures (Rabinowitz et al., 2005), and the number of migratory neural precursors and proliferative cells increased over time after collagen matrix implantation following TBI in rats (Huang et al., 2012). Collagen matrices can also enhance recovery from injury states: implantation of collagen scaffolds without growth factors or stem cell seeding in rats after surgical trauma decreased lesion volume and improved neurological function, assessed by tactile adhesive removal test and modified neurological severity scores (Huang et al., 2012).
While many similarities exist between different neural injury models, there is definite difference between the mechanism of injury in TBI compared to surgical trauma. Unlike surgical trauma where tissue is simply severed, TBI induces destruction by much more complex injury by sudden compressive force. Specifically, there is injury to the tissue by direct impact (contusion) as well as white matter degeneration distant from the site of impact (Hall et al., 2008). The mechanism of injury is different both on the macroscopic as well as cellular point of view. Although the prior studies showing neuroregenerative benefits of collagen matrix in surgical injury has been promising, its utility in TBI has not been explored thus far. The ability of biological scaffolds to promote tissue repair has been extensively studied in the past. Biological scaffolds such as porcine urinary bladder ECM have chemoattractant and mitogenic properties (Beattie et al., 2009; Reing et al., 2009). Studies using ECM in neural tissues found differences in the material properties as well as neurotrophic and chemotactic properties among ECMs from different sources (Crapo et al, 2012; Medberry et al., 2013). ECMs applied to the CNS can differentiate neural stem cells into neurons (Crapo et al., unpublished data) and increase neurite length (Medberry et al., 2013) compared to an ECM from urinary bladder. In stroke injury, an ECM scaffold also allowed for survival and integration of neural stem cells in the lesion cavity (Bible et al., 2012), as well as reduced lesion volume and neurobehavioral recovery (Zhang et al., 2013). These studies suggest that the possible mechanism of functional recovery and reduced lesion volume seen in our study is due to migration and/or preservation of neural precursor cells. With increased neuronal growth into the damaged site of contusion injury, functional deficits of hippocampal and cortical tissue may be attenuated, leading to improvements in spatial learning memory and reduced lesion volume. The ability of collagen to stimulate neurogenesis and improve neural growth, differentiation, and survival is likely multifactorial. Collagen itself has a chemotactic property as it is actively degraded, attracting fibroblasts and promoting fast colonization (Postlethwaite et al., 1978). Since no collagen matrix in its original form was visualized at the time of dissection in our experiments, it has likely degraded over time with the byproducts of this degradation promoting recovery in perilesional areas. Another possibility is the integration into the brain parenchyma. The high porosity of graft is thought to aid in fibroblast migration and vas-
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cularization of the dural graft, hastening the integration of graft with the native tissue (Narotam et al., 1995). Collagen has also been shown to enhance the survival of CNS neurons and suppress apoptosis in culture (Carri et al., 1992; O’Connor et al., 2000). However, it is unknown whether these are the particular mechanisms that resulted in the functional improvement and reduction of lesion volume noted in our study. Our results show that lesion volume and cell survival is enhanced after implantation of collagen matrix. However, there was no difference in GFAP and collagen staining (Figs. 5, 6) at 3 week time point. Further investigation with various cell types including neurons and microglia and a timeline of histology is needed to verify the specific mechanism. This study demonstrates for the first time that clinically used collagen matrix common in neurosurgical settings promotes hippocampal cell survival, reduces lesion volume, and protects behavioral outcome in a rat model of TBI. There are several limitations in our study design. The implantation of the matrix occurred immediately following injury, which may differ from a clinically relevant scenario. In a setting of human TBI, application of a neuroprotective scaffold may not occur until hours after initial injury when patients are brought into the clinical setting. However, the intention of this experiment is to first verify the clinical benefit after injury to the brain parenchyma. In the future studies, implantation at various time points will ensue. Also, the control group in this study does not have an inert space occupying material in place of collagen matrix. Another limitation is that a specific molecular mechanism underlying these improvements has not been elucidated. In the future studies, the mechanism of neuroprotection by collagen matrix after TBI will be further investigated. Levels of neurotrophic factors such as GDNF and BDNF, as well as the progress of neuronal migration over several time points following collagen matrix implantation will be closely observed. Implantation of the matrix at different timeframes after injury will also be studied, since post injury intervention will happen at any time between hours to days after injury in a realistic setting for human TBI.
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of CA1 and CA3 hippocampal neurons. Thus, collagen matrix has neuroprotective effect shown by behavioral and histological data after TBI. As future ideas for utilizing biomatrix for treatment of TBI develop further, combination of regenerative strategies such as behavioral training along with cell based therapies may have additional benefit (Dunkerson et al., 2014). Acknowledgments The authors would like to thank Dr. Xiecheng Ma for her help with animal preparation and injury and Sherman Culver and Megan Sullivan for their help with animal behavioral testing. This work was supported by NIH/NINDS and by the Pittsburgh Foundation Walter L. Copeland Fund. References Bakshi, A., Fisher, O., Dagci, T., Himes, B.T., Fischer, I., & Lowman, A. (2004). Mechanically engineered hydrogel scaffolds for axonal growth and angiogenesis after transplantation in spinal cord injury. J Neurosurg, Spine 1, 322-329. Beattie, A.J., Gilbert, T.W., Guyot, J.P., Yates, A.J., & Badylak, S.F. (2009). Chemoattraction of progenitor cells by remodeling extracellular matrix scaffolds. Tissue Eng, 15(Pt A), 1119-1125. Bible, E., Dell’Acqua, F., Solanky, B., Balducci, A., Crapo, P.M., Badylak, S.F., Ahrens, E.T., & Modo, M. (2012). Non-invasive imaging of transplanted human neural stem cells and ECM scaffold remodeling in the stroke-damaged rat brain by (19)F- and diffusion-MRI. Biomaterials, 33, 2858-2871. Brazda, N., & Muller, H.W. (2009). Pharmacological modification of the extracellular matrix to promote regeneration of the injured brain and spinal cord. Progress Brain Res, 175, 269-281. Carri, N.G., Rubin, K., Gullberg, D., & Ebendal, T. (1992). Neuritogenesis on collagen substrates. Involvement of integrin-like matrix receptors in retinal fibre outgrowth on collagen. Int J Dev Neurosci, 10, 393-405. Crapo, P.M., Medberry, C.J., Reing, J.E., Tottey, S., van der Merwe, Y., Jones, K.E., & Badylak, S.F. (2012). Biologic scaffolds composed of central nervous system extracellular matrix. Biomaterials, 33, 3539-3547. Dixon, C.E., Clifton, G.L., Lighthall, J.W., Yaghmai, A.A., & Hayes, R.L. (1991). A controlled cortical impact model of traumatic brain injury in the rat. J Neurosci Meth, 39, 253-262.
5. Conclusions
Dixon, C.E., Hamm, R.J., Taft, W.C., & Hayes, R.L. (1994). Increased anticholinergic sensitivity following closed skull impact and controlled cortical impact traumatic brain injury in the rat. J Neurotraum, 11, 275-287.
After treatment of TBI rats with collagen matrix at the site of injury, there is improvement in spatial memory as well as reduced lesion volume and reduced loss
Dixon, C.E., Kraus, M.F., Kline, A.E., Ma, X., Yan, H.Q., Griffith, R.G., Wolfson, B.M., & Marion, D.W. (1999). Amantadine improves water maze performance without affecting motor behavior following traumatic brain injury in rats. Restor Neurol Neuros, 14, 285-294.
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Dunkerson, J., Moritz, K. E., Young, J., Pionk, T., Fink, K., Rossignol, J., Smith, J.S., et al. (2014). Combining enriched environment and induced pluripotent stem cell therapy results in improved cognitive and motor function following traumatic brain injury. Restor Neurol Neurosci, 32(5), 675-687. doi: 10.3233/RNN-140408 Exo, J.L., Shellington, D.K., Bayir, H., Vagni, V.A., JanescoFeldman, K., Ma, L., Hsia, C.J., Clark, R.S., Jenkins, L.W., Dixon, C.E., & Kochanek, P.M. (2009). Resuscitation of traumatic brain injury and hemorrhagic shock with polynitroxylated albumin, hextend, hypertonic saline, and lactated Ringer’s: Effects on acute hemodynamics, survival, and neuronal death in mice. J Neurotraum, 26, 2403-2408. Giacino, J.T., Whyte, J., Bagiella, E., Kalmar, K., Childs, N., Khademi, A., Eifert, B., Long, D., Katz, D.I., Cho, S., Yablon, S.A., Luther, M., Hammond, F.M., Nordenbo, A., Novak, P., Mercer, W., Maurer-Karattup, P., & Sherer, M. (2012). Placebocontrolled trial of amantadine for severe traumatic brain injury. New Eng J Med, 366, 819-826. Hamm, R.J., Dixon, C.E., Gbadebo, D.M., Singha, A.K., Jenkins, L.W., Lyeth, B.G., & Hayes, R.L. (1992). Cognitive deficits following traumatic brain injury produced by controlled cortical impact. J Neurotraum, 9, 11-20. Huang, K.F., Hsu, W.C., Chiu, W.T., & Wang, J.Y. (2012). Functional improvement and neurogenesis after collagen-GAG matrix implantation into surgical brain trauma. Biomaterials, 33, 20672075. Kumar, A., & Loane, D.J. (2012). Neuroinflammation after traumatic brain injury: Opportunities for therapeutic intervention. Brain Behav Immun, 26, 1191-1201. Labour, M.N., Banc, A., Tourrette, A., Cunin, F., Verdier, J.M., Devoisselle, J.M., Marcilhac, A., & Belamie, E. (2012). Thick collagen-based 3D matrices including growth factors to induce neurite outgrowth. Acta Biomater, 8, 3302-3312. Lu, D., Mahmood, A., Qu, C., Hong, X., Kaplan, D., & Chopp, M. (2007). Collagen scaffolds populated with human marrow stromal cells reduce lesion volume and improve functional outcome after traumatic brain injury. Neurosurgery, 61, 596-602; discussion 602-593. Matsuse, D., Kitada, M., Ogura, F., Wakao, S., Kohama, M., Kira, J., Tabata, Y., & Dezawa, M. (2011). Combined transplantation of bone marrow stromal cell-derived neural progenitor cells with a collagen sponge and basic fibroblast growth factor releasing microspheres enhances recovery after cerebral ischemia in rats. Tissue Eng, 17(Pt A), 1993-2004. Medberry, C.J., Crapo, P.M., Siu, B.F., Carruthers, C.A., Wolf, M.T., Nagarkar, S.P., Agrawal, V., Jones, K.E., Kelly, J., Johnson, S.A., Velankar, S.S., Watkins, S.C., Modo, M., & Badylak, S.F. (2013). Hydrogels derived from central nervous system extracellular matrix. Biomaterials, 34, 1033-1040. Morris, R. (1984). Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Meth, 11, 47-60. Narotam, P.K., van Dellen, J.R., & Bhoola, K.D. (1995). A clinicopathological study of collagen sponge as a dural graft in neurosurgery. J Neurosurg, 82, 406-412. Nordblom, J., Persson, J. K., Aberg, J., Blom, H., Engqvist, H., Brismar, H., Mattsson, P., et al. (2012). FGF1 containing
biodegradable device with peripheral nerve grafts induces corticospinal tract regeneration and motor evoked potentials after spinal cord resection. Restor Neurol Neurosci, 30(2), 91-102. doi: 10.3233/RNN-2011-0623 O’Connor, S.M., Stenger, D.A., Shaffer, K.M., Maric, D., Barker, J.L., & Ma, W. (2000). Primary neural precursor cell expansion, differentiation and cytosolic Ca(2+) response in three-dimensional collagen gel. J Neurosci Meth, 102, 187-195. Park, K.I., Teng, Y.D., & Snyder, E.Y. (2002). The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Nat Biotech, 20, 1111-1117. Postlethwaite, A.E., Seyer, J.M., & Kang, A.H. (1978). Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides. P Nat Acad Sci U S A, 75, 871-875. Rabinowitz, L., Monnerie, H., Shashidhara, S., & Le Roux, P.D. (2005). Growth of rat cortical neurons on DuraGen, a collagenbased dural graft matrix. Neurol Res, 27, 887-894. Reing, J.E., Zhang, L., Myers-Irvin, J., Cordero, K.E., Freytes, D.O., Heber-Katz, E., Bedelbaeva, K., McIntosh, D., Dewilde, A., Braunhut, S.J., & Badylak, S.F. (2009). Degradation products of extracellular matrix affect cell migration and proliferation. Tissue Eng, 15(Pt A), 605-614. Scheff, S.W., Baldwin, S.A., Brown, R.W., & Kraemer, P.J. (1997). Morris water maze deficits in rats following traumatic brain injury: Lateral controlled cortical impact. J Neurotraum, 14, 615-627. Shellington, D.K., Du, L., Wu, X., Exo, J., Vagni, V., Ma, L., JaneskoFeldman, K., Clark, R.S., Bayir, H., Dixon, C.E., Jenkins, L.W., Hsia, C.J., & Kochanek, P.M. (2011). Polynitroxylated pegylated hemoglobin: A novel neuroprotective hemoglobin for acute volume-limited fluid resuscitation after combined traumatic brain injury and hemorrhagic hypotension in mice. Crit Care Med, 39, 494-505. Singleton, R.H., Yan, H.Q., Fellows-Mayle, W., & Dixon, C.E. (2010). Resveratrol attenuates behavioral impairments and reduces cortical and hippocampal loss in a rat controlled cortical impact model of traumatic brain injury. J Neurotraum, 27, 1091-1099. Tate, M.C., Shear, D.A., Hoffman, S.W., Stein, D.G., Archer, D.R., & LaPlaca, M.C. (2002). Fibronectin promotes survival and migration of primary neural stem cells transplanted into the traumatically injured mouse brain. Cell Transplant, 11, 283295. Woerly, S., Marchand, R., & Lavallee, C. (1990). Intracerebral implantation of synthetic polymer/biopolymer matrix: A new perspective for brain repair. Biomaterials, 11, 97-107. Xiong, Y., Qu, C., Mahmood, A., Liu, Z., Ning, R., Li, Y., Kaplan, D.L., Schallert, T., & Chopp, M. (2009). Delayed transplantation of human marrow stromal cell-seeded scaffolds increases transcallosal neural fiber length, angiogenesis, and hippocampal neuronal survival and improves functional outcome after traumatic brain injury in rats. Brain Res, 1263, 183-191. Yu, H., Cao, B., Feng, M., Zhou, Q., Sun, X., Wu, S., Jin, S., Liu, H., & Lianhong, J. (2010). Combinated transplantation of neural stem cells and collagen type I promote functional recovery after cerebral ischemia in rats. Anat Rec, 293, 911-917.
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Restorative Neurology and Neuroscience 33 (2015) 95–104 DOI 10.3233/RNN-140430 IOS Press
Neuroprotective effects of collagen matrix in rats after traumatic brain injury Samuel S. Shina,b,c , Ramesh Grandhib , Jeremy Henchira,b , Hong Q. Yana,b,c , Stephen F. Badylakd and C. Edward Dixona,b,c,e,∗ a Brain
Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania of Neurological Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania c Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, Pennsylvania d McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania e Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania b Department
Abstract. Purpose: In previous studies, collagen based matrices have been implanted into the site of lesion in different models of brain injury. We hypothesized that semisynthetic collagen matrix can have neuroprotective function in the setting of traumatic brain injury. Methods: Rats were subjected to sham injury or controlled cortical impact. They either received extracellular matrix graft (DuraGen) over the injury site or did not receive any graft and underwent beam balance/beam walking test at post injury days 1–5 and Morris water maze at post injury days 14–18. Animals were sacrificed at day 18 for tissue analysis. Results: Collagen matrix implantation in injured rats did not affect motor function (beam balance test: p = 0.627, beam walking test: p = 0.921). However, injured group with collagen matrix had significantly better spatial memory acquisition (p < 0.05). There was a significant reduction in lesion volume, as well as neuronal loss in CA1 (p < 0.001) and CA3 (p < 0.05) regions of the hippocampus in injured group with collagen matrix (p < 0.05). Conclusions: Collagen matrix reduces contusional lesion volume, neuronal loss, and cognitive deficit after traumatic brain injury. Further studies are needed to demonstrate the mechanisms of neuroprotection by collagen matrix. Keywords: Collagen, matrix, traumatic brain injury, bioscaffold, neuroregeneration, neurorepair
1. Introduction Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Long-term neurobehavioral deficits are common among survivors of TBI, but only a few pharmacological agents such as Amantadine have shown success in improving neurobehavioral function after TBI (Giacino et al., 2012). Strategies to increase neuroregeneration (Brazda and Muller, 2009) ∗ Corresponding
author: C. Edward Dixon, PhD, Department of Neurological Surgery, University of Pittsburgh, 201 Hill Building, 3434 Fifth Avenue, Pittsburgh, PA 15260, USA. Tel.: +1 412 383 2188; Fax: +1 412 624 0943; E-mail: [email protected].
and reduce inflammation (Kumar and Loane, 2012) after traumatic injury to the central nervous system (CNS) have been previously reviewed. However, clinical treatment options for TBI are limited due to the heterogeneity of the disease and the irreversibility of injury to neural structures. DuraGen (DuraGen Plus, DP-1011) is a semisynthetic Type I collagen matrix from bovine deep flexor tendon with delicate mechanical properties. Although collagen matrices such as DuraGen have been commonly used for dural repair, several recent studies have explored the use of collagen matrices and biological scaffold ECMs in repair of neural injury. Collagen
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matrices have been used as a platform for therapeutic delivery of stem cells in experimental models of ischemia (Matsuse et al., 2011; Park et al., 2002), spinal cord injury (Bakshi et al., 2004), and TBI (Lu et al., 2007; Tate et al., 2002; Xiong et al., 2009). These matrices have been shown to provide an effective foundation by which stem cells can anchor after tissue injury, leading to improved cell survival, neovascularization, and functional recovery. Biodegradable materials releasing growth factors have been utilized in the past to improve nerve regeneration (Nordblom et al., 2012). Even without seeding of neuronal cells or addition of factors such as brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), or fibronectin (FN), implantation of collagen composite hydrogels into adult rat brains had penetration by neural tissue (Woerly et al., 1990). Urinary bladder matrix (UBM) derived from porcine bladder also contains collagen, fibronectin, and various growth factors. In a recent study, injection of UBM into the injury site after TBI reduced both lesion volume and white matter injury (Zhang et al., 2013). In addition, there was an improvement in vestibulomotor function. In fact, recent study has shown that even implantation of collagen-based scaffolds alone can lead to increased levels of GDNF and BDNF, as well as proliferation and migration of neural precursor cells after brain injury (Huang et al., 2012). The current study aimed to determine if there is a beneficial effect of a readily available, FDA-approved collagen matrix graft in the injury site after TBI in rats, and the results demonstrated that collagen matrix may reduce the degree of injury and induce functional deficits.
2. Materials and methods 2.1. Ethics statement All animal work in this study has been conducted according to ethical national/international guidelines. The experiments in this study have been approved by Institutional Animal Care and Use Committee of the University of Pittsburgh.
light:dark cycle and given food and water ad libitum. Rats were divided into 4 groups: TBI rats with collagen matrix graft (IC), sham rats with collagen matrix graft (SC), TBI rats with no graft (IN), and sham rats with no graft (SN). For tissue staining studies, 39 animals were used: IC = 10, IN = 9, SN = 9, and SC = 11. For behavioral experiments, 48 animals were used: IC = 13, IN = 11, SN = 13, and SC = 11. 2.3. Surgical procedures for controlled cortical impact (CCI) Animals were injured by Controlled Cortical Impact (CCI) as previously described (Dixon et al., 1991). Briefly, rats were anesthetized with 5% isoflurane (IsoFlo; Abbott Laboratories, North Chicago, IL), carried in a 2:1 ratio of N2 O/O2 , endotracheally intubated, and maintained on a small-animal ventilator (Harvard Rodent Ventilator, Model 683; Harvard Apparatus, Holliston, MA) with 2% isoflurane in the same carrier gas mixture. Body temperature was maintained at 37◦ C. After placement in a stereotactic frame (David Kopf Instruments, Tujunga, CA), a parasagittal craniectomy 8 mm in diameter was performed, and TBI was induced with a 6-mm in diameter beveled impactor tip (impact speed: 4 m/s, injury depth: 2.6 mm, dwell time: 50 msec). The CCI device produced cortical contusion 8 mm in diameter. For IC and SC animals, a piece of collagen matrix (DuraGen, Integra LifeSciences Corporation, Plainsboro, NJ) about 8 mm in diameter was placed over the lesion site just after injury and entirely covering the craniotomy site. CCI at this injury severity, tears the dura, thus allowing direct contact of the collagen matrix with injured cortical tissue. The collagen matrix used in this study did not contain any chemo-attractants or growth factors, The exposed area was then closed by suturing the scalp while holding the 0.3 mm thick piece of matrix firmly against the injury site. For IN and SN animals, no graft material was used at the lesion site. Rats were allowed to recover from anesthesia before returning to their housing facility. In sham rats (SN, SC), craniectomy without CCI was performed. 2.4. Behavioral experiments: Motor test
2.2. Animals Eighty-seven male Sprague-Dawley rats (Harlan Laboratories Inc., Indianapolis, IN, USA) weighing 280–300 g were used. Rats were housed in 12:12 hour
On days 1–5 post injury, motor functions were tested using beam balance and beam walking tests, as previously described using our standard behavioral protocol (Singleton et al., 2010). Each of the four groups
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consisted of n = 10–13 animals. For beam balance test, rats were placed on a suspended wooden beam (width: 1.5 cm), and their latency to stay on the beam was recorded up to maximum of 60 seconds. Rats were assessed prior to injury, and then tested for 5 consecutive days after injury. For beam walking test, the latency of travel across a narrow wooden beam (length: 100 cm, width: 2.5 cm) to move away from loud white noise and bright environment into a darkened goal box at the end of the beam was measured. There are four metal pegs (diameter: 3 mm, height: 4 cm) to challenge the coordination and agility of rats during the travel. Two days before injury, rats have been trained to traverse the beam. For 5 days after injury, latency of travel was recorded up to maximum of 60 seconds. All motor and cognitive testing was performed blinded to experimental group designation. 2.5. Behavioral experiments: Spatial memory test A variant of the Morris water maze (MWM) (Morris et al., 1984) was employed as described previously on days 14–18 after injury (Dixon et al., 1994, 1999; Hamm et al., 1992; Scheff et al., 1997) in the same rats that had undergone motor testing. The water maze used a 120 cm-diameter and 60cmhigh plastic pool filled with 25◦ C water to a depth of 28 cm. A platform 10 cm in diameter and 27 cm high, 1 cm below the water’s surface, was used as the hidden goal platform. The pool was located in a 2.5 × 2.5 m room with numerous extramaze cues that remained constant during testing. A video tracking system (AnyMaze, Stoelting, Inc. Wood Dale, IL) recorded and quantitated the swimming motions of the rats. The hidden platform version of the MWM was used to assess spatial memory acquisition. Fiveday acquisition blocks consisted of four daily trials over 5 consecutive days. Each of the four groups consisted of n = 10–13 animals. Rats started a trial from each of the four possible start locations in random order. The location of the platform was held constant for each animal and was varied between animals. Rats were given a maximum of 120 seconds to find the hidden platform. All rats were allowed to remain on the platform for 30 seconds before being placed in a heated incubator between trials (4-minute inter-trial interval). A single probe trial to determine memory retention was performed on day 5 of MWM testing, in which the platform was removed and the percentage time in the platform quadrant and swim
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speed was measured for 60 sec by the video tracking system. 2.6. Tissue preparation, lesion volume analysis, hippocampal cell counting After completion of behavioral tests, rats were sacrificed with an overdose of sodium pentobarbital (100 mg/kg, intraperitoneal) before delivering 0.9% saline then 10% phosphate-buffered formalin intracardially. Brain tissue was dissected and post fixed in 10% phosphate buffered formalin for 5 days. The forebrain was paraffin-embedded and sectioned using a microtome (Leitz 1512; Leica Microsystems, Germany) at 7 m thickness every 500 m. The sections were then de-paraffinized, hydrated, and stained with hematoxylin and eosin (H & E) and coverslipped for lesion volume analysis. Using a light microscope, coronal sections of rat brain tissue were analyzed with imaging software (Image J version 1.46r, National Institutes of Health, Bethesda, MD, USA). Contusion volume was outlined on each section and lesion volume was calculated using Cavalieri principle. Surviving neurons in CA1 and CA3 regions of hippocampi were counted as described previously (Exo et al., 2009; Shellington et al., 2011). The number of neurons was counted on H & E stained sections by a blinded observer using light microscopy. Glial fibrillary acidic protein (GFAP) and trichrome stains were performed for the (Figs. 5, 6). 2.7. Methods for GFAP immunofluorescence staining on ECM sections Whole frontal brains were embedded in paraffin and then sectioned at 5m. Sections were mounted on SuperFrost Plus microscope slides and allowed to dry for 24 hours or more. Deparaffinization of the sections was done with 3 washes of xylene for 5 minutes each. Sections were then hydrated with 2 washes of 100% EtOH and 2 washes of 95% EtOH for 3 minutes each. Slides then were washed for 3 minutes in 1× TBS Automation Wash Buffer (BioCare Medical #TWB945 M) and then 3 minutes in distilled water. Antigen retrieval was then performed using BioCare Medical #CB910 M Antigen Decloaker by 5 minutes of medium power heating in a microwave followed by a 20 minute cooling off period. Once the antigen decloaker has cooled, a 5 minute distilled water wash followed by a 5 minute Automation Buffer wash was performed. The slides were then incubated at room
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temperature for 30 minutes with 10% Normal (donkey, Sigma #D9663) Blocking Serum. Slides were then incubated at 4◦ C overnight with GFAP (Millipore #MAB360) diluted in 5% Normal Blocking Serum. On the following day, the slides were allowed to acclimate to room temperature for 15 minutes followed by 2 washes for 10 minutes each in Automation Buffer. Slides were then incubated for 1 hour at room temperature with the secondary antibody (Life Technologies #A-21203). After incubation of the secondary antibody, 3 washes of 5 minutes each were performed followed by coverslipping with Vectashield Hardset.
volumes of each group were compared using Student’s T test. Morris water maze probe trial data and hippocampal neuronal counts were analyzed using one-way analysis of variance (ANOVA). When a significant difference between the groups was found by one-way ANOVA, post hoc analysis was performed using Student-Newman-Keuls method. Motor testing and Morris water maze results were analyzed using repeated measures ANOVA and Student-NewmanKeuls post hoc analysis.
3. Results 2.8.. Methods for masson trichrome stain After whole frontal brains were embedded in paraffin then sectioned at 5m, sections were mounted on SuperFrost Plus microscope slides and allowed to dry for 24 hours or more. We used the NovaUltra Masson Trichrome Stain Kit from IHCWorld (#IW3006). Deparaffinization of the sections was done with 2 washes with xylene for 10 minutes each. Slides were hydrated with 2 washes of 100% EtOH for 5 minutes followed by washes in 95% EtOH and 70% EtOH for 1 minute each. Slides were then rinsed in distilled tap water, and then post-fixed in Bouin’s Solution for 1 hour in a 56◦ C oven. Slides then were rinsed with running tap water for 5–10 minutes, and then stained with Weigert’s Iron Hematoxylin Solution for 10 minutes. Following Weigert’s Hematoxylin, the slides were rinsed in running tap water for 5–10 minutes and then washed in distilled water. Slides were then stained with Biebrich Scarlet-Acid Fuchsin Solution for 10–15 minutes, and then washed in distilled water. Slides were then differentiated with PP Acid Solution for 10–15 minutes and then transferred directly into Aniline Blue Solution for 5–10 minutes. Slides were then rinsed briefly in distilled and differentiated in Acetic Acid Solution for 2–5 minutes. Following a wash in distilled water, the slides were dehydrated with 1 wash of 95% EtOH for 1 minute and then 2 washes of 100% EtOH for 1 minute each. Slides were then washed in xylene twice for 5 minutes each, and then coverslipped with Permount (Fisher Scientific #SP-15). 2.9. Statistical analysis Statistical testing was performed using SPSS software (v. 20, SPSS Inc., Chicago, IL, USA). Lesion
Repeated measures ANOVA showed significant differences in group (F3,47 = 21.796, p < 0.001) and day (F5,235 = 23.812, p < 0.001) comparisons on beam balance testing on days 1–5 (Fig. 1a), as well as group × day interaction (F15,235 = 6.721, p < 0.001). Post hoc analysis showed that although both injured groups were significantly different from both sham groups (p < 0.001), there was no significant difference between the IC and IN groups (p = 0.627). In beam walking test (Fig. 1b), there were significant differences in group (F3,47 = 78.404, p < 0.001), day (F3,47 = 70.900, p < 0.001), and group x day interaction (F15,235 = 9.479, p < 0.001). Similar to beam balance testing, both the injured groups were significantly different from both sham groups (p < 0.001), but there was no significant difference between IC and IN groups (p = 0.921). Spatial working memory was assessed using MWM at a later time point on days 14–18 (Fig. 2). Time course of latency to find the platform for 5 consecutive days was used to assess spatial learning. Repeated measures ANOVA showed group (F3,44 = 14.076, p < 0.001) and day (F4,176 = 27.798, p < 0.001) differences. However, there was no group x day interaction (F12,176 = 1.377, p = 0.181). Post hoc analysis revealed significant reduction in latency in IC group compared to IN group (p < 0.05) as well as significant differences between SN and SC groups compared to IN group (p < 0.01). Spatial memory retention was assessed by using probe trials, which showed significant group differences (F3,44 = 3.616, p = 0.02). By post hoc analysis, there was a significant difference between duration spent in target quadrant by IN (18.0 ± 1.6 seconds) and SN (25.5 ± 2.1 seconds) groups (p < 0.05), showing injury effect on spatial memory retention. However, there was no significant difference between IC and
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Fig. 1. Motor function. The motor data was assessed by beam balance test (A) and beam walking test (B). In both experiments, IC and IN were significantly different from SN (∗ p < 0.001).
Fig. 2. Spatial working memory assessed by Morris water maze test. There is a significant improvement in the latency of the injured rats after collagen matrix treatment (A) (∗ p < 0.05, ∗∗ p < 0.01 compared to IN). Probe trial showed decreased duration spent in target quadrant in injured animals (∗ p < 0.05), but no effect of collagen matrix on memory retention (B).
IN groups (p = 0.81), indicating no effect of collagen matrix on memory retention after injury. Also, there were no significant differences among groups for swim speed, indicating that the performance differences in water maze were not due to physical disability (F3,44 = 0.744, p = 0.53). The average swim speed were as follows: IC = 0.269 ± 0.008, IN = 0.269 ± 0.011, SN = 0.278 ± 0.011, SC = 0.287 ± 0.007. After sacrifice of the rats, tissues were analyzed using light microscopic morphometry. Lesion volume comparisons showed a significant reduction in injuryinduced lesion volume (p < 0.05) in IC rats compared to
IN rats (Fig. 3). The degree of injury was also assessed by counting the number of neurons in CA1 and CA3 regions of the hippocampus (Fig. 4). For CA1 neuronal counts, there was a significant between group difference (F3,35 = 28.611, p < 0.001). There was a significant reduction in both IN and IC groups’ neurons compared to SN (p < 0.001). Also, collagen matrix was effective in attenuating neuronal losses: IC compared to IN showed significant difference (p < 0.001). For CA3 neuronal counts, there was also a significant between group difference (F3,35 = 18.551, p < 0.001). The IC and IN groups showed significant reduction
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Fig. 3. Tissue lesion after injury. The histological samples were observed after no treatment (A), or collagen matrix treatment (B). Quantified lesion volume is also presented (C). There was significant reduction of lesion size in injured animals that received collagen matrix (∗ p < 0.05).
Fig. 4. Hippocampal neuronal counts. There was a significant decrease in the number of neurons in injured groups in both CA1 (A) and CA3 (B) regions. Collagen matrix treatment significantly attenuated this neuronal loss compared to untreated injured controls. (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).
in the number of surviving neurons compared to the SN group (p < 0.01 and p < 0.001, respectively). Collagen matrix also attenuated the neuronal loss in CA3: IC compared to IN showed significant difference (p < 0.05). Several other studies were performed to further understand the effect of collagen matrix. Frozen sections were stained for glial fibrillary acidic
protein (GFAP), showing no difference in IC and IN in terms of astrocytes in the pericontusional areas (Fig. 5). Also, in order to visualize whether collagen matrix was still present or not, trichrome staining was performed showing no evidence of remaining collagen matrix after sacrifice at 3 weeks (Fig. 6).
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Fig. 5. GFAP staining through the injured region shows no increase in the number of astrocytes among TBI rats treated with collagen matrix (Left) compared to TBI rats with no treatment (Right).
Fig. 6. Two sections through the hippocampus of injured rats treated with collagen matrix show no residual matrix overlying the injured region on trichrome stain.
4. Discussion In this study, we applied the collagen-based matrix to the area of cortical injury of rats after trauma. This treatment in injured rats resulted in improvement of spatial memory acquisition, and histological sections
revealed reduced lesion volume as well as increased hippocampal neuronal counts. These histological sections showed no remaining collagen matrix at the site of injury. Thus, its neuroprotective effect in TBI may be due to its activity as biological scaffold ECMs enhancing repair of neural injury rather than repairing the
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dural defect. In fact, the surgical procedure in this study is not a dural repair procedure, since it entailed implantation of collagen matrix at a width of an injury cavity. Collagen matrix implanted was only large enough to fit into the injury cavity following TBI, and did not cover overlap into the intact parts of the dura as a dural repair procedure would have. Future studies are needed to elucidate the mechanisms by which collagen matrix conferred benefit in the present study. Most studies involving the use of biological scaffold matrices in experimental brain injury models have employed a strategy in which stem cells have been implanted in the local environment with the addition of exogenous trophic factors. Impregnation of growth factors within the ECM or delivery of trophic factors by microspheres optimizes the microenvironment for neural growth and stimulates neurogenesis, proliferation, migration, and neural survival. Collagen matrices with impregnated nerve growth factor (NGF) and BDNF can enhance neurite outgrowth (Labour et al., 2012), and microspheres that release basic fibroblast growth factor (FGF2) can improve cell survival and behavioral recovery after cerebral ischemia (Matsuse et al., 2011). In addition, the implantation of a collagen matrix can increase BDNF and GDNF (Huang et al., 2012), leading to cell proliferation and functional recovery. The use of collagen as a scaffold, without the concomitant presence of trophic factors, has been shown to stabilize and optimize the survival and differentiation of neuronal stem cells after local transplantation (Woerly et al., 1990; Huang et al., 2012; Yu et al., 2010). Importantly, collagen matrix by itself may result in neural growth and functional recovery. Acellular and denaturized collagen matrices have been shown to promote neural growth (Zhang et al., 2013; Huang et al., 2012; Rabinowitz et al., 2005), showing that these matrices alone may be biologically active. An In vitro study of collagen matrix has demonstrated the ability of the synthetic dural matrix to support growth of axons and dendrites in neuronal cultures (Rabinowitz et al., 2005), and the number of migratory neural precursors and proliferative cells increased over time after collagen matrix implantation following TBI in rats (Huang et al., 2012). Collagen matrices can also enhance recovery from injury states: implantation of collagen scaffolds without growth factors or stem cell seeding in rats after surgical trauma decreased lesion volume and improved neurological function, assessed by tactile adhesive removal test and modified neurological severity scores (Huang et al., 2012).
While many similarities exist between different neural injury models, there is definite difference between the mechanism of injury in TBI compared to surgical trauma. Unlike surgical trauma where tissue is simply severed, TBI induces destruction by much more complex injury by sudden compressive force. Specifically, there is injury to the tissue by direct impact (contusion) as well as white matter degeneration distant from the site of impact (Hall et al., 2008). The mechanism of injury is different both on the macroscopic as well as cellular point of view. Although the prior studies showing neuroregenerative benefits of collagen matrix in surgical injury has been promising, its utility in TBI has not been explored thus far. The ability of biological scaffolds to promote tissue repair has been extensively studied in the past. Biological scaffolds such as porcine urinary bladder ECM have chemoattractant and mitogenic properties (Beattie et al., 2009; Reing et al., 2009). Studies using ECM in neural tissues found differences in the material properties as well as neurotrophic and chemotactic properties among ECMs from different sources (Crapo et al, 2012; Medberry et al., 2013). ECMs applied to the CNS can differentiate neural stem cells into neurons (Crapo et al., unpublished data) and increase neurite length (Medberry et al., 2013) compared to an ECM from urinary bladder. In stroke injury, an ECM scaffold also allowed for survival and integration of neural stem cells in the lesion cavity (Bible et al., 2012), as well as reduced lesion volume and neurobehavioral recovery (Zhang et al., 2013). These studies suggest that the possible mechanism of functional recovery and reduced lesion volume seen in our study is due to migration and/or preservation of neural precursor cells. With increased neuronal growth into the damaged site of contusion injury, functional deficits of hippocampal and cortical tissue may be attenuated, leading to improvements in spatial learning memory and reduced lesion volume. The ability of collagen to stimulate neurogenesis and improve neural growth, differentiation, and survival is likely multifactorial. Collagen itself has a chemotactic property as it is actively degraded, attracting fibroblasts and promoting fast colonization (Postlethwaite et al., 1978). Since no collagen matrix in its original form was visualized at the time of dissection in our experiments, it has likely degraded over time with the byproducts of this degradation promoting recovery in perilesional areas. Another possibility is the integration into the brain parenchyma. The high porosity of graft is thought to aid in fibroblast migration and vas-
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cularization of the dural graft, hastening the integration of graft with the native tissue (Narotam et al., 1995). Collagen has also been shown to enhance the survival of CNS neurons and suppress apoptosis in culture (Carri et al., 1992; O’Connor et al., 2000). However, it is unknown whether these are the particular mechanisms that resulted in the functional improvement and reduction of lesion volume noted in our study. Our results show that lesion volume and cell survival is enhanced after implantation of collagen matrix. However, there was no difference in GFAP and collagen staining (Figs. 5, 6) at 3 week time point. Further investigation with various cell types including neurons and microglia and a timeline of histology is needed to verify the specific mechanism. This study demonstrates for the first time that clinically used collagen matrix common in neurosurgical settings promotes hippocampal cell survival, reduces lesion volume, and protects behavioral outcome in a rat model of TBI. There are several limitations in our study design. The implantation of the matrix occurred immediately following injury, which may differ from a clinically relevant scenario. In a setting of human TBI, application of a neuroprotective scaffold may not occur until hours after initial injury when patients are brought into the clinical setting. However, the intention of this experiment is to first verify the clinical benefit after injury to the brain parenchyma. In the future studies, implantation at various time points will ensue. Also, the control group in this study does not have an inert space occupying material in place of collagen matrix. Another limitation is that a specific molecular mechanism underlying these improvements has not been elucidated. In the future studies, the mechanism of neuroprotection by collagen matrix after TBI will be further investigated. Levels of neurotrophic factors such as GDNF and BDNF, as well as the progress of neuronal migration over several time points following collagen matrix implantation will be closely observed. Implantation of the matrix at different timeframes after injury will also be studied, since post injury intervention will happen at any time between hours to days after injury in a realistic setting for human TBI.
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of CA1 and CA3 hippocampal neurons. Thus, collagen matrix has neuroprotective effect shown by behavioral and histological data after TBI. As future ideas for utilizing biomatrix for treatment of TBI develop further, combination of regenerative strategies such as behavioral training along with cell based therapies may have additional benefit (Dunkerson et al., 2014). Acknowledgments The authors would like to thank Dr. Xiecheng Ma for her help with animal preparation and injury and Sherman Culver and Megan Sullivan for their help with animal behavioral testing. This work was supported by NIH/NINDS and by the Pittsburgh Foundation Walter L. Copeland Fund. References Bakshi, A., Fisher, O., Dagci, T., Himes, B.T., Fischer, I., & Lowman, A. (2004). Mechanically engineered hydrogel scaffolds for axonal growth and angiogenesis after transplantation in spinal cord injury. J Neurosurg, Spine 1, 322-329. Beattie, A.J., Gilbert, T.W., Guyot, J.P., Yates, A.J., & Badylak, S.F. (2009). Chemoattraction of progenitor cells by remodeling extracellular matrix scaffolds. Tissue Eng, 15(Pt A), 1119-1125. Bible, E., Dell’Acqua, F., Solanky, B., Balducci, A., Crapo, P.M., Badylak, S.F., Ahrens, E.T., & Modo, M. (2012). Non-invasive imaging of transplanted human neural stem cells and ECM scaffold remodeling in the stroke-damaged rat brain by (19)F- and diffusion-MRI. Biomaterials, 33, 2858-2871. Brazda, N., & Muller, H.W. (2009). Pharmacological modification of the extracellular matrix to promote regeneration of the injured brain and spinal cord. Progress Brain Res, 175, 269-281. Carri, N.G., Rubin, K., Gullberg, D., & Ebendal, T. (1992). Neuritogenesis on collagen substrates. Involvement of integrin-like matrix receptors in retinal fibre outgrowth on collagen. Int J Dev Neurosci, 10, 393-405. Crapo, P.M., Medberry, C.J., Reing, J.E., Tottey, S., van der Merwe, Y., Jones, K.E., & Badylak, S.F. (2012). Biologic scaffolds composed of central nervous system extracellular matrix. Biomaterials, 33, 3539-3547. Dixon, C.E., Clifton, G.L., Lighthall, J.W., Yaghmai, A.A., & Hayes, R.L. (1991). A controlled cortical impact model of traumatic brain injury in the rat. J Neurosci Meth, 39, 253-262.
5. Conclusions
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After treatment of TBI rats with collagen matrix at the site of injury, there is improvement in spatial memory as well as reduced lesion volume and reduced loss
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