http://informahealthcare.com/bij ISSN: 0269-9052 (print), 1362-301X (electronic) Brain Inj, Early Online: 1–8 ! 2015 Informa UK Ltd. DOI: 10.3109/02699052.2015.1018325
ORIGINAL ARTICLE
Widespread microglial activation in patients deceased from traumatic brain injury Antonio Vela´zquez1, Marisa Ortega1,2, Santiago Rojas1, Francisco Javier Gonza´lez-Oliva´n2, & Alfonso Rodrı´guez-Baeza1 Departamento de Ciencias Morfolo´gicas, Facultad de Medicina, Universitat Auto`noma de Barcelona, Barcelona, Spain and 2Instituto de Medicina Legal de Catalun˜a, Barcelona, Spain
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Abstract
Keywords
Primary objective: The role of microglial activation in traumatic brain injury (TBI) has been extensively described in established animal models. In contrast, very few studies have analysed this process in human patients, the majority being focused on the local reaction in the contused parenchyma. In this work, the main objective was the analysis of microglial activation in brain regions distant from the primary lesion. Research design: Morphological changes of microglia were evaluated in the cerebral cortex of patients deceased from TBI in comparison with control subjects. Methods and procedures: Cortical samples from five cases with TBI and 10 controls were evaluated using Ricinus communis lectin histochemistry and conventional Hematoxylin-eosin staining. Main outcomes and results: It was observed that microglial cells from patients with TBI presented shorter and thicker cellular projections compared with controls. Moreover, the percentage of histological area reactive to lectin was statistically higher in samples from subjects with TBI. These signs of microglial activation were observed in all of the analysed cortical areas, thus indicating a generalized effect on the whole cerebral cortex. The results are consistent with previous imaging PET studies performed in living patients with the 11C-PK11195 radiotracer. Conclusions: The findings indicate that TBI induces a widespread activation of brain microglia which affects all cortical areas, including those distant from the contusion site.
Human, microglial activation, traumatic brain injury, Ricinus communis lectin
Introduction Traumatic brain injury (TBI) is a leading cause of permanent neurological disability in young adults, with an associated mortality rate of up to 40% [1]. In addition, TBI is considered a risk factor for the development of neurodegenerative diseases such as Alzheimer’s disease [2]. After the primary lesion, caused by cerebral contusion, very often the damage injury commonly extends to other areas of the brain. Several physiopathological processes have been implicated in the development of secondary brain damage after TBI, including inflammatory response, disruption of the blood–brain barrier, excitotoxicity, mitochondrial failure, oxidative stress, astrogliosis and microglial activation [1]. Microglial cells are the resident macrophages of the central nervous system (CNS) [3]. Currently, the general consensus is
Correspondence: Professor Alfonso Rodrı´guez-Baeza, Unit of Human Anatomy, Medicine Faculty, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Tel: +34935811948. E-mail: [email protected]
History Received 5 August 2014 Accepted 9 January 2015 Published online 11 June 2015
that they are members of the mononuclear lineage that invade the CNS during neurodevelopment [4]. The resident microglial cells change their morphology under pathological conditions, such as encephalic trauma or ischaemia, and become activated macrophages in the injured brain parenchyma [5]. Additionally, new waves of circulating monocytes are recruited by the action of different adhesion molecules expressed in the vascular endothelium [6]. These migrating monocytes enter injured brain regions and become indistinguishable from the resident activated microglia [7]. The role of these activated microglia/macrophages in acute CNS lesions could be dual. On the one hand, they produce reactive oxygen and nitrogen species, metalloproteinases and other molecules that exacerbate the damage in the surrounding and initially preserved tissue [8, 9]. Moreover, they express molecules that promote neuronal apoptosis [10] and secrete cytokines, such as TNF- and IL-1 , which induce brain inflammation, disruption of the blood–brain barrier, vascular oedema formation and the recruitment of more inflammatory cells [11]. On the other hand, activated microglia/macrophages can secrete neurotrophic factors and participate in the
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development of glial scar delimiting the damaged tissue [12]. They also eliminate damaged myelin and tissue debris, this way preventing the induction of secondary cell death in the surrounding tissue [13]. Moreover, it has been reported that some populations of microglia can secrete anti-inflammatory cytokines, inducing a negative feedback and limiting the expansion of inflammatory reaction [14]. In summary, the results reported in the literature show that microglial/macrophages are the main effectors of inflammatory response in CNS [15], having a negative effect in most cases, although some of their functions could also be beneficial for lesion outcome [16]. Few studies have specifically analysed microglial activation in human patients with TBI. Moreover, these studies only evaluated microglial activation in the focus of contusion as well as the surrounding tissue, not in other brain areas. Electron microscopy of brain samples from patients with TBI revealed amoeboid phagocytic cells in perivascular spaces and parenchyma of the injured area [17]. Other studies report the use of immunohistochemical analysis of specific antigens to evaluate the temporal evolution of microglial activation in TBI. They found that, 1–2 days after contusion, expression of CD14 was increased in the site of the lesion as well as the surrounding areas. This antigen is expressed in activated macrophages and circulating monocytes, but not in resident microglia. The expression peak of CD14 was observed 4–8 days after injury and remained high for several weeks [18]. Similar results were reported for other macrophage-related proteins, such as MRP8, MRP14 and the proliferating antigen MIB-1, which were not expressed until 72 hours post-injury [19]. Additionally, the quantification of CD68, major complex of histocompatibility (MCHII), leukocyte common antigen (LCA) and HAM56 supported the delayed activation of microglial cells in TBI [20]. Previous Positron Emission Tomography (PET) studies performed with the 11C-PK11195 radiotracer analysed microglial activation in vivo in patients that had suffered severe TBI. Very interestingly, these studies showed that microglial activation remains active several months after the time of injury and, more importantly, showed a widespread distribution of microglial activation throughout the brain [21, 22]. The hypothesis that a focal TBI could induce global changes in encephalic microglia has not been sufficiently explored in humans. This work seeks to analyse microglial activation in the different brain regions from patients that had died from severe TBI, with special attention to those areas not initially damaged by the contusion.
Materials and methods
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parahippocampal–hippocampal region. In subjects with TBI, an additional sample of the contused area was also collected. Histological processing Cortical samples were fixed by immersion in Zamboni’s fixative for 24 hours, rinsed in PBS and stored in 70% ethanol at 4 C until use. Each sample was processed in parallel for lectin histochemistry and Hematoxylin-Eosin (H-E) staining using standard protocols. In brief, sections of 50 mm from fixed brain samples were obtained with a vibratome and maintained in Olmos cryoprotective solution at 20 C until use. Sections were rinsed in PBS, the endogenous peroxidase was blocked and incubated in free floating conditions with Ricinus comunis lectin coupled to biotin. A second step with streptavidin coupled to horseradish peroxidase was used to amplify the signal and, finally, the sections were developed with diaminobenzidine. After that, the obtained sections were placed in gelatin-coated slides, dehydrated and definitively mounted with DPX for its evaluation. Another part of the each sample was dehydrated and embedded in paraffin for histological evaluation. Sections of 10 mm were obtained, collected in gelatin-coated slides and stained following standard H-E protocol. Before its examination, slides were dehydrated and mounted with DPX. Analysis of microglial activation Images were obtained with a Nikon Eclipse E-1000 microscope (Nikon, Japan) and a digital camera (CC-12, Soft Imaging System GmbH, Munich, Germany). Qualitative analysis of the sections stained with Ricinus communis lectin was performed to evaluate the morphology of the microglial cells. Quantitative densitometric analysis of Ricinus communis lectin staining was performed with ImageJÕ 1,47v software (National Institutes of Health, Bethesda, MD, USA). Micrographs were captured at a magnification of 200. The percentage of area positively stained was measured in three fields selected randomly in the cortical grey matter of each section. The mean of these values was calculated and used for the statistical analysis. Analysis of vacuolation Analysis of vacuolation was conducted through the visual count of vacuoles of three randomly selected fields of H-E staining micrographs captured at a magnification of 200. The mean of obtained values was calculated and used for the statistical analysis.
Subjects and samples
Statistical analysis
Post-mortem brain tissue was obtained at the Instituto Anato´mico Forense de Barcelona (currently Institut de Medicina Legal de Catalunya) just after medico-legal autopsy before the application of law 14/2007 (BOE 159 of 4 July 2007) and in agreement with the 2nd transitory disposition of the law. Cortical samples of 1.5 1.5 0.5 cm were collected from five patients who died as result of a severe TBI and from 10 control subjects (Table I). Samples were taken from frontal, parietal, occipital, temporal lobes and the
The analysis was an unmatched cases and controls study. The Gaussian distribution of the data was determined with ShapiroWilk test. Variables that followed a normal distribution were analysed with two-way multiple interactions analysis of variance (MANOVA) followed by Bonferroni correction. Mann-Whitney U-test and Kruskal-Wallis test were used for those variables that did not follow Gaussian distribution. All analyses were performed with the computer software R CommanderÕ 2.14.2 (http://www.rcommander.com/).
Widespread microglial activation in deceased TBI patients
DOI: 10.3109/02699052.2015.1018325
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Table I. Subjects included in the study.
Reference
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TBI T1 T2 T3 T4 T5 Control C1 C2 C3 C4 C5 C6 C7 C8 C9 C10
Age
Post-mortem delay (hours)
Time of survival after the TBI (days)
Bifrontal and left temporal TBI. Orbitofrontal and temporal TBI. Temporal TBI. TBI; Subdural Haemorrhage. Bifrontal TBI, Bleeding Subarachnoid.
12 25 24 24 22
4 12 3 7 2
Cardiac sudden death Cardiac sudden death Drowned Suicide Cervical fracture Overdose of drugs Suicide Abdominal trauma Overdose of drugs Thoracic damage
13 8 5 15 20 5 18 22 7 6
– – – – – – – – – –
Sex
Death cause
68 86 71 70 16
Male Male Female Female Male
68 59 55 41 75 30 42 20 41 27
Male Male Male Male Female Male Male Male Male Male
Results Population characteristics For control subjects, mean age was 45.8 years (range ¼ 20–68 years) and for patients with TBI it was 62.2 years (range ¼ 16–86 years). Twelve subjects were male and three female. The post-mortem time elapsed from death to necropsy was 15.06 ± 7.56 hours (21.4 ± 5.37 hours in patients with TBI and 11.9 ± 6, 54 hours in control patients). The survival time after brain contusion in patients with TBI was 5.6 ± 4.04 days (Table I). Qualitative analysis of microglial morphology Visual inspection of the slices showed that microglial cells from cases with TBI had thicker and shorter cellular projections compared to controls (Figure 1). Different degrees of microglial activation were identified between the different patients with TBI (Figure 1). In contrast, similar microglial morphology was observed in the different brain regions obtained from the same patient (Figure 2). In some of the cases with TBI, rounded activated macrophages were observed in the contused areas (Figure 2(B)). In the control group, the aspect of microglia was similar in all areas evaluated, presenting the typical morphology of resident microglia with very thin and long cellular processes (Figure 1(G)). No clear differences were observed between control subjects. The microglial morphology found in samples from brains with TBI was not detected in any control sample (Figure 1). Percentage of area stained with lectin Statistical analysis showed that percentages of histologic area reactive to lectin were significantly higher in the sections obtained from cases with TBI compared with those from controls (p50.001) (Figure 3). These differences affected all brain regions evaluated, but were particularly significant in frontal lobes (p ¼ 0.000509; F ¼ 21.04; df ¼ 1), the frontomedial regions (p ¼ 0.000426; F ¼ 21.95; df ¼ 1), occipital areas (p ¼ 0.000024; F ¼ 40.78; df ¼ 1), parietal (p ¼ 0.000146; F ¼ 27.99; df ¼ 1) and temporal lobes
(p ¼ 0.00000132; F ¼ 70.4; df ¼ 1). This difference between controls and patients with TBI was a little less pronounced in the parahippocampal–hippocampal region, but still statistically significant (p ¼ 0.00476; F ¼ 1155; df ¼ 1). Tissue vacuolation The histological evaluation of tissue vacuolation showed a highly heterogeneous pattern between subjects. In fact, the area of cellular vacuolation was variable among the different samples analysed, with no obvious relation with TBI (Figure 4). Statistical analysis of vacuolation did not show any significant results among the different cases and controls in any anatomic region assessed (Mann-Whitney U test; p40.05) with the exception of the parahippocampal– hippocampal region (p ¼ 0.02773) (Figure 5).
Discussion The results show that the cerebral cortex of patients who died as a consequence of a TBI has a generalized activation of the microglia, not restricted to the area of contusion. It is hypothesized that this microglial activation could induce a pro-inflammatory environment in the undamaged cerebral parenchyma. The findings are consistent with previous imaging PET studies, performed in living patients with the radiotracer 11C-PK11195 [21, 22]. In contrast to microglial activation, the extent of tissue vacuolation was similar between controls and patients with TBI, in all brain regions, with the exception of the parahippocampus-hippocampus. Tissue vacuolation may occur as a result of cytotoxic or vasogenic oedema. However, it could also be caused by post mortem autolysis. In fact, the degree of vacuolation was not homogeneous between subjects of the same group, thus indicating that both cause of death as well as differences in time until autopsy could contribute to a variable degree of vacuolation in the samples and potentially hide the presence of subtle changes between controls and cases with TBI. Alternatively, it is possible that vacuolation could be especially prevalent and localized in the affected brain areas and practically inexistent in distant areas,
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Figure 1. Micrographs of microglia cells obtained from three controls (A, C, E) and three patients with TBI (B, D, F). Morphological differences of the microglia between subjects with TBI (H) and controls (G) are clearly appreciated at high power field.
despite the occurrence of microglial alterations. Interestingly, parahippocampus-hippocampus is the only region that showed a significantly higher degree of vacuolation in patients with TBI. This may be due to the higher susceptibility of the hippocampus to insults that occur during the clinical course of cerebral contusion, such as excitotoxicity. Microglial activation has been extensively characterized in animal models of TBI [23–25]. In these models, a limited and localized necrotic focus is induced by applying a noxious agent. The microglial cells surrounding this area become activated and migrate progressively to remove all damaged tissue. These animal models have been used to establish the dynamics of microglial activation and their relation with the
evolution of the lesion. Moreover, they have proven useful to explore the role of activated microglia in secondary neuronal injury and to evaluate different therapeutic interventions [26–28]. In this way, it has been suggested that treatments capable of reducing microglial activation could improve the functional outcome after encephalic contusion [29]. However, there are several significant differences between animal models and patients affected by TBI. In animal models of TBI, the lesion is induced by applying a focal injury in a determined cortical area that hardly affects the remainder brain regions, at least not to the level observed in deceased patients. Moreover, animals generally survive this lesion, do not suffer coma and usually preserve their capacity to eat,
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DOI: 10.3109/02699052.2015.1018325
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Figure 2. Micrographs of microglia cells obtained from different areas of the cortex of the patient (T5) showing a widespread cortex microglial activation. The images correspond to: temporal cortex (A), frontal lobe (B), occipital lobe—that is the area of contusion in this case (C), fronto-medial region (D), parietal lobe (E) and parahippocampal–hippocampal region (F).
Figure 3. Percentages of area stained with Ricinus communis of each selected anatomical region of subjects with TBI and controls (***p50.001; **p50.01).
drink and breathe without artificial support. In contrast, human patients may suffer TBI in any encephalic area and the occurrence of multiple contusions is common. As a result, findings observed in animal models are not completely comparable with those found in patients who die from TBI. Microglial cells, like other macrophages, may acquire differentiated phenotypes during their activation. Polarization to one phenotype or another depends on the stimulus present in their local microenvironment. In this way, LPS and IFN promote classic activation known as M1, which produces elevated levels of pro-inflammatory cytokines and toxic metabolites. In contrast, IL-4 and IL-13 cytokines induce alternative phenotypic activation or M2, which promotes angiogenesis, tissue repair and remodelling of the extracellular matrix and suppresses destructive immune responses [30]. As a result, it could be speculated that the activated M1 phenotype would be deleterious in initially non-damaged brain regions, whereas activation to the M2 phenotype in the contusion area would have positive effects. Further studies designed to characterize the predominant phenotypes of activated microglia in TBI could contribute to shed light on their specific physiopathological role. It would
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Figure 4. Micrographs of H-E staining obtained in controls (A, C, E, G) and patients with TBI (B, D, F, H).
be especially relevant to correlate the pattern of microglial activation with the changes observed in neuroradiology studies. This approach is out of the scope and aims of the present work. However, additional studies to explore microglial activation and phenotypical differentiation, in connection with the occurrence of brain oedema or haemorrhagic transformation, are necessary to precisely characterize the specific contribution of microglial sub-populations and their activation with the evolution of TBI. Taking into consideration the deleterious effects of M1 macrophages on other cell types [8–11], it is hypothesized that this generalized microglial activation could induce
neuronal death and disruption of the blood–brain barrier in regions not affected by the initial traumatism. Thus, generalized activation of microglia in the cerebral cortex could hamper brain homeostasis and could contribute to worsening the prognosis of patients with TBI. As such, inhibiting the generalized microglial activation would constitute a promising therapeutic approach to limit the secondary cerebral damage. The evaluation of this new therapeutic target could be supported by the in vivo use of PET radiotracers directed against the peripheral benzodiazepine receptors, such as 11 C-PK11195. Additionally, using this imaging technology to identify microglial activation in areas distant from contusion
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DOI: 10.3109/02699052.2015.1018325
Figure 5. Average of vacuole number in observed in H-E sections (*p50.05).
could pave the way to understand the significance of this process and its contribution to the pathogenesis and prognosis of TBI.
Conclusions TBI induces a widespread activation of brain microglia that affects even the cortical areas distant from the contusion.
Acknowledgments The study is financially supported by Fondo de Investigaciones Sanitarias Instituto Carlos III (01/0008-01).
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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ORIGINAL ARTICLE
Widespread microglial activation in patients deceased from traumatic brain injury Antonio Vela´zquez1, Marisa Ortega1,2, Santiago Rojas1, Francisco Javier Gonza´lez-Oliva´n2, & Alfonso Rodrı´guez-Baeza1 Departamento de Ciencias Morfolo´gicas, Facultad de Medicina, Universitat Auto`noma de Barcelona, Barcelona, Spain and 2Instituto de Medicina Legal de Catalun˜a, Barcelona, Spain
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Abstract
Keywords
Primary objective: The role of microglial activation in traumatic brain injury (TBI) has been extensively described in established animal models. In contrast, very few studies have analysed this process in human patients, the majority being focused on the local reaction in the contused parenchyma. In this work, the main objective was the analysis of microglial activation in brain regions distant from the primary lesion. Research design: Morphological changes of microglia were evaluated in the cerebral cortex of patients deceased from TBI in comparison with control subjects. Methods and procedures: Cortical samples from five cases with TBI and 10 controls were evaluated using Ricinus communis lectin histochemistry and conventional Hematoxylin-eosin staining. Main outcomes and results: It was observed that microglial cells from patients with TBI presented shorter and thicker cellular projections compared with controls. Moreover, the percentage of histological area reactive to lectin was statistically higher in samples from subjects with TBI. These signs of microglial activation were observed in all of the analysed cortical areas, thus indicating a generalized effect on the whole cerebral cortex. The results are consistent with previous imaging PET studies performed in living patients with the 11C-PK11195 radiotracer. Conclusions: The findings indicate that TBI induces a widespread activation of brain microglia which affects all cortical areas, including those distant from the contusion site.
Human, microglial activation, traumatic brain injury, Ricinus communis lectin
Introduction Traumatic brain injury (TBI) is a leading cause of permanent neurological disability in young adults, with an associated mortality rate of up to 40% [1]. In addition, TBI is considered a risk factor for the development of neurodegenerative diseases such as Alzheimer’s disease [2]. After the primary lesion, caused by cerebral contusion, very often the damage injury commonly extends to other areas of the brain. Several physiopathological processes have been implicated in the development of secondary brain damage after TBI, including inflammatory response, disruption of the blood–brain barrier, excitotoxicity, mitochondrial failure, oxidative stress, astrogliosis and microglial activation [1]. Microglial cells are the resident macrophages of the central nervous system (CNS) [3]. Currently, the general consensus is
Correspondence: Professor Alfonso Rodrı´guez-Baeza, Unit of Human Anatomy, Medicine Faculty, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Tel: +34935811948. E-mail: [email protected]
History Received 5 August 2014 Accepted 9 January 2015 Published online 11 June 2015
that they are members of the mononuclear lineage that invade the CNS during neurodevelopment [4]. The resident microglial cells change their morphology under pathological conditions, such as encephalic trauma or ischaemia, and become activated macrophages in the injured brain parenchyma [5]. Additionally, new waves of circulating monocytes are recruited by the action of different adhesion molecules expressed in the vascular endothelium [6]. These migrating monocytes enter injured brain regions and become indistinguishable from the resident activated microglia [7]. The role of these activated microglia/macrophages in acute CNS lesions could be dual. On the one hand, they produce reactive oxygen and nitrogen species, metalloproteinases and other molecules that exacerbate the damage in the surrounding and initially preserved tissue [8, 9]. Moreover, they express molecules that promote neuronal apoptosis [10] and secrete cytokines, such as TNF- and IL-1 , which induce brain inflammation, disruption of the blood–brain barrier, vascular oedema formation and the recruitment of more inflammatory cells [11]. On the other hand, activated microglia/macrophages can secrete neurotrophic factors and participate in the
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development of glial scar delimiting the damaged tissue [12]. They also eliminate damaged myelin and tissue debris, this way preventing the induction of secondary cell death in the surrounding tissue [13]. Moreover, it has been reported that some populations of microglia can secrete anti-inflammatory cytokines, inducing a negative feedback and limiting the expansion of inflammatory reaction [14]. In summary, the results reported in the literature show that microglial/macrophages are the main effectors of inflammatory response in CNS [15], having a negative effect in most cases, although some of their functions could also be beneficial for lesion outcome [16]. Few studies have specifically analysed microglial activation in human patients with TBI. Moreover, these studies only evaluated microglial activation in the focus of contusion as well as the surrounding tissue, not in other brain areas. Electron microscopy of brain samples from patients with TBI revealed amoeboid phagocytic cells in perivascular spaces and parenchyma of the injured area [17]. Other studies report the use of immunohistochemical analysis of specific antigens to evaluate the temporal evolution of microglial activation in TBI. They found that, 1–2 days after contusion, expression of CD14 was increased in the site of the lesion as well as the surrounding areas. This antigen is expressed in activated macrophages and circulating monocytes, but not in resident microglia. The expression peak of CD14 was observed 4–8 days after injury and remained high for several weeks [18]. Similar results were reported for other macrophage-related proteins, such as MRP8, MRP14 and the proliferating antigen MIB-1, which were not expressed until 72 hours post-injury [19]. Additionally, the quantification of CD68, major complex of histocompatibility (MCHII), leukocyte common antigen (LCA) and HAM56 supported the delayed activation of microglial cells in TBI [20]. Previous Positron Emission Tomography (PET) studies performed with the 11C-PK11195 radiotracer analysed microglial activation in vivo in patients that had suffered severe TBI. Very interestingly, these studies showed that microglial activation remains active several months after the time of injury and, more importantly, showed a widespread distribution of microglial activation throughout the brain [21, 22]. The hypothesis that a focal TBI could induce global changes in encephalic microglia has not been sufficiently explored in humans. This work seeks to analyse microglial activation in the different brain regions from patients that had died from severe TBI, with special attention to those areas not initially damaged by the contusion.
Materials and methods
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parahippocampal–hippocampal region. In subjects with TBI, an additional sample of the contused area was also collected. Histological processing Cortical samples were fixed by immersion in Zamboni’s fixative for 24 hours, rinsed in PBS and stored in 70% ethanol at 4 C until use. Each sample was processed in parallel for lectin histochemistry and Hematoxylin-Eosin (H-E) staining using standard protocols. In brief, sections of 50 mm from fixed brain samples were obtained with a vibratome and maintained in Olmos cryoprotective solution at 20 C until use. Sections were rinsed in PBS, the endogenous peroxidase was blocked and incubated in free floating conditions with Ricinus comunis lectin coupled to biotin. A second step with streptavidin coupled to horseradish peroxidase was used to amplify the signal and, finally, the sections were developed with diaminobenzidine. After that, the obtained sections were placed in gelatin-coated slides, dehydrated and definitively mounted with DPX for its evaluation. Another part of the each sample was dehydrated and embedded in paraffin for histological evaluation. Sections of 10 mm were obtained, collected in gelatin-coated slides and stained following standard H-E protocol. Before its examination, slides were dehydrated and mounted with DPX. Analysis of microglial activation Images were obtained with a Nikon Eclipse E-1000 microscope (Nikon, Japan) and a digital camera (CC-12, Soft Imaging System GmbH, Munich, Germany). Qualitative analysis of the sections stained with Ricinus communis lectin was performed to evaluate the morphology of the microglial cells. Quantitative densitometric analysis of Ricinus communis lectin staining was performed with ImageJÕ 1,47v software (National Institutes of Health, Bethesda, MD, USA). Micrographs were captured at a magnification of 200. The percentage of area positively stained was measured in three fields selected randomly in the cortical grey matter of each section. The mean of these values was calculated and used for the statistical analysis. Analysis of vacuolation Analysis of vacuolation was conducted through the visual count of vacuoles of three randomly selected fields of H-E staining micrographs captured at a magnification of 200. The mean of obtained values was calculated and used for the statistical analysis.
Subjects and samples
Statistical analysis
Post-mortem brain tissue was obtained at the Instituto Anato´mico Forense de Barcelona (currently Institut de Medicina Legal de Catalunya) just after medico-legal autopsy before the application of law 14/2007 (BOE 159 of 4 July 2007) and in agreement with the 2nd transitory disposition of the law. Cortical samples of 1.5 1.5 0.5 cm were collected from five patients who died as result of a severe TBI and from 10 control subjects (Table I). Samples were taken from frontal, parietal, occipital, temporal lobes and the
The analysis was an unmatched cases and controls study. The Gaussian distribution of the data was determined with ShapiroWilk test. Variables that followed a normal distribution were analysed with two-way multiple interactions analysis of variance (MANOVA) followed by Bonferroni correction. Mann-Whitney U-test and Kruskal-Wallis test were used for those variables that did not follow Gaussian distribution. All analyses were performed with the computer software R CommanderÕ 2.14.2 (http://www.rcommander.com/).
Widespread microglial activation in deceased TBI patients
DOI: 10.3109/02699052.2015.1018325
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Table I. Subjects included in the study.
Reference
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TBI T1 T2 T3 T4 T5 Control C1 C2 C3 C4 C5 C6 C7 C8 C9 C10
Age
Post-mortem delay (hours)
Time of survival after the TBI (days)
Bifrontal and left temporal TBI. Orbitofrontal and temporal TBI. Temporal TBI. TBI; Subdural Haemorrhage. Bifrontal TBI, Bleeding Subarachnoid.
12 25 24 24 22
4 12 3 7 2
Cardiac sudden death Cardiac sudden death Drowned Suicide Cervical fracture Overdose of drugs Suicide Abdominal trauma Overdose of drugs Thoracic damage
13 8 5 15 20 5 18 22 7 6
– – – – – – – – – –
Sex
Death cause
68 86 71 70 16
Male Male Female Female Male
68 59 55 41 75 30 42 20 41 27
Male Male Male Male Female Male Male Male Male Male
Results Population characteristics For control subjects, mean age was 45.8 years (range ¼ 20–68 years) and for patients with TBI it was 62.2 years (range ¼ 16–86 years). Twelve subjects were male and three female. The post-mortem time elapsed from death to necropsy was 15.06 ± 7.56 hours (21.4 ± 5.37 hours in patients with TBI and 11.9 ± 6, 54 hours in control patients). The survival time after brain contusion in patients with TBI was 5.6 ± 4.04 days (Table I). Qualitative analysis of microglial morphology Visual inspection of the slices showed that microglial cells from cases with TBI had thicker and shorter cellular projections compared to controls (Figure 1). Different degrees of microglial activation were identified between the different patients with TBI (Figure 1). In contrast, similar microglial morphology was observed in the different brain regions obtained from the same patient (Figure 2). In some of the cases with TBI, rounded activated macrophages were observed in the contused areas (Figure 2(B)). In the control group, the aspect of microglia was similar in all areas evaluated, presenting the typical morphology of resident microglia with very thin and long cellular processes (Figure 1(G)). No clear differences were observed between control subjects. The microglial morphology found in samples from brains with TBI was not detected in any control sample (Figure 1). Percentage of area stained with lectin Statistical analysis showed that percentages of histologic area reactive to lectin were significantly higher in the sections obtained from cases with TBI compared with those from controls (p50.001) (Figure 3). These differences affected all brain regions evaluated, but were particularly significant in frontal lobes (p ¼ 0.000509; F ¼ 21.04; df ¼ 1), the frontomedial regions (p ¼ 0.000426; F ¼ 21.95; df ¼ 1), occipital areas (p ¼ 0.000024; F ¼ 40.78; df ¼ 1), parietal (p ¼ 0.000146; F ¼ 27.99; df ¼ 1) and temporal lobes
(p ¼ 0.00000132; F ¼ 70.4; df ¼ 1). This difference between controls and patients with TBI was a little less pronounced in the parahippocampal–hippocampal region, but still statistically significant (p ¼ 0.00476; F ¼ 1155; df ¼ 1). Tissue vacuolation The histological evaluation of tissue vacuolation showed a highly heterogeneous pattern between subjects. In fact, the area of cellular vacuolation was variable among the different samples analysed, with no obvious relation with TBI (Figure 4). Statistical analysis of vacuolation did not show any significant results among the different cases and controls in any anatomic region assessed (Mann-Whitney U test; p40.05) with the exception of the parahippocampal– hippocampal region (p ¼ 0.02773) (Figure 5).
Discussion The results show that the cerebral cortex of patients who died as a consequence of a TBI has a generalized activation of the microglia, not restricted to the area of contusion. It is hypothesized that this microglial activation could induce a pro-inflammatory environment in the undamaged cerebral parenchyma. The findings are consistent with previous imaging PET studies, performed in living patients with the radiotracer 11C-PK11195 [21, 22]. In contrast to microglial activation, the extent of tissue vacuolation was similar between controls and patients with TBI, in all brain regions, with the exception of the parahippocampus-hippocampus. Tissue vacuolation may occur as a result of cytotoxic or vasogenic oedema. However, it could also be caused by post mortem autolysis. In fact, the degree of vacuolation was not homogeneous between subjects of the same group, thus indicating that both cause of death as well as differences in time until autopsy could contribute to a variable degree of vacuolation in the samples and potentially hide the presence of subtle changes between controls and cases with TBI. Alternatively, it is possible that vacuolation could be especially prevalent and localized in the affected brain areas and practically inexistent in distant areas,
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Figure 1. Micrographs of microglia cells obtained from three controls (A, C, E) and three patients with TBI (B, D, F). Morphological differences of the microglia between subjects with TBI (H) and controls (G) are clearly appreciated at high power field.
despite the occurrence of microglial alterations. Interestingly, parahippocampus-hippocampus is the only region that showed a significantly higher degree of vacuolation in patients with TBI. This may be due to the higher susceptibility of the hippocampus to insults that occur during the clinical course of cerebral contusion, such as excitotoxicity. Microglial activation has been extensively characterized in animal models of TBI [23–25]. In these models, a limited and localized necrotic focus is induced by applying a noxious agent. The microglial cells surrounding this area become activated and migrate progressively to remove all damaged tissue. These animal models have been used to establish the dynamics of microglial activation and their relation with the
evolution of the lesion. Moreover, they have proven useful to explore the role of activated microglia in secondary neuronal injury and to evaluate different therapeutic interventions [26–28]. In this way, it has been suggested that treatments capable of reducing microglial activation could improve the functional outcome after encephalic contusion [29]. However, there are several significant differences between animal models and patients affected by TBI. In animal models of TBI, the lesion is induced by applying a focal injury in a determined cortical area that hardly affects the remainder brain regions, at least not to the level observed in deceased patients. Moreover, animals generally survive this lesion, do not suffer coma and usually preserve their capacity to eat,
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DOI: 10.3109/02699052.2015.1018325
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Figure 2. Micrographs of microglia cells obtained from different areas of the cortex of the patient (T5) showing a widespread cortex microglial activation. The images correspond to: temporal cortex (A), frontal lobe (B), occipital lobe—that is the area of contusion in this case (C), fronto-medial region (D), parietal lobe (E) and parahippocampal–hippocampal region (F).
Figure 3. Percentages of area stained with Ricinus communis of each selected anatomical region of subjects with TBI and controls (***p50.001; **p50.01).
drink and breathe without artificial support. In contrast, human patients may suffer TBI in any encephalic area and the occurrence of multiple contusions is common. As a result, findings observed in animal models are not completely comparable with those found in patients who die from TBI. Microglial cells, like other macrophages, may acquire differentiated phenotypes during their activation. Polarization to one phenotype or another depends on the stimulus present in their local microenvironment. In this way, LPS and IFN promote classic activation known as M1, which produces elevated levels of pro-inflammatory cytokines and toxic metabolites. In contrast, IL-4 and IL-13 cytokines induce alternative phenotypic activation or M2, which promotes angiogenesis, tissue repair and remodelling of the extracellular matrix and suppresses destructive immune responses [30]. As a result, it could be speculated that the activated M1 phenotype would be deleterious in initially non-damaged brain regions, whereas activation to the M2 phenotype in the contusion area would have positive effects. Further studies designed to characterize the predominant phenotypes of activated microglia in TBI could contribute to shed light on their specific physiopathological role. It would
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Figure 4. Micrographs of H-E staining obtained in controls (A, C, E, G) and patients with TBI (B, D, F, H).
be especially relevant to correlate the pattern of microglial activation with the changes observed in neuroradiology studies. This approach is out of the scope and aims of the present work. However, additional studies to explore microglial activation and phenotypical differentiation, in connection with the occurrence of brain oedema or haemorrhagic transformation, are necessary to precisely characterize the specific contribution of microglial sub-populations and their activation with the evolution of TBI. Taking into consideration the deleterious effects of M1 macrophages on other cell types [8–11], it is hypothesized that this generalized microglial activation could induce
neuronal death and disruption of the blood–brain barrier in regions not affected by the initial traumatism. Thus, generalized activation of microglia in the cerebral cortex could hamper brain homeostasis and could contribute to worsening the prognosis of patients with TBI. As such, inhibiting the generalized microglial activation would constitute a promising therapeutic approach to limit the secondary cerebral damage. The evaluation of this new therapeutic target could be supported by the in vivo use of PET radiotracers directed against the peripheral benzodiazepine receptors, such as 11 C-PK11195. Additionally, using this imaging technology to identify microglial activation in areas distant from contusion
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DOI: 10.3109/02699052.2015.1018325
Figure 5. Average of vacuole number in observed in H-E sections (*p50.05).
could pave the way to understand the significance of this process and its contribution to the pathogenesis and prognosis of TBI.
Conclusions TBI induces a widespread activation of brain microglia that affects even the cortical areas distant from the contusion.
Acknowledgments The study is financially supported by Fondo de Investigaciones Sanitarias Instituto Carlos III (01/0008-01).
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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