CALCITONIN PUMP IMPROVES NERVE REGENERATION AFTER TRANSECTION INJURY AND REPAIR JI-GENG YAN, MD, PhD, JOHN LOGIUDICE, MD, JOHN DAVIS, BS, LIN-LING ZHANG, MD, MICHAEL AGRESTI, MS, JAMES SANGER, MD, HANI S. MATLOUB, MD, and ROBERT HAVLIK, MD Department of Plastic Surgery, Medical College of Wisconsin, 8700 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA Accepted 6 May 2014 ABSTRACT: Introduction: After nerve injury, excessive calcium impedes nerve regeneration. We previously showed that calcitonin improved nerve regeneration in crush injury. We aimed to validate the direct effect of calcitonin on transected and repaired nerve. Methods: Two rat groups (n 5 8) underwent sciatic nerve transection followed by direct repair. In the calcitonin group, a calcitonin-filled mini-osmotic pump was implanted subcutaneously, with a catheter parallel to the repaired nerve. The control group underwent repair only, without a pump. Evaluation and comparison between the groups included: (1) compound muscle action potential recording of the extensor digitorum longus (EDL) muscle; (2) tetanic muscle force test of EDL; (3) nerve calcium concentration; and (4) nerve fiber count and calcified spot count. Results: The calcitonin pump group showed superior recovery. Conclusions: Calcitonin affects injured and repaired peripheral nerve directly. The calcitonin-filled miniosmotic pump improved nerve functional recovery by accelerating calcium absorption from the repaired nerve. This finding has potential clinical applications. Muscle Nerve 51: 229–234, 2015
Calcium ions play a crucial role in normal neuronal function.1,2 Intraneural scarring is ubiquitous after peripheral nerve injury, which damages numerous membrane transporters, including plasma membrane calcium ATPase (PMCA) and sodium calcium exchanger (NCX). PMCA and NCX normally act as the main mechanisms of calcium extrusion to maintain a much lower intracellular concentration of calcium relative to the extracellular concentration. The large calcium concentration gradient is necessary for a wide variety of cell signaling and transduction events. However, when PMCA and NCX are damaged, calcium influx via diffusion then overwhelms the calcium extrusion mechanisms.3,4 The resultant rise in intracellular calcium can disrupt normal cell signaling and transductions and can activate secondary Ca21-dependent cascades, such as apoptosis, resulting in Schwann cell death.5 There have not been any studies attempting to validate the theory that excessive calcium impedes nerve regeneration Abbreviations: AM, acetoxy methyl; BNB, blood–nerve barrier; CMAP, compound muscle action potential; CS, calcified spot; EDL, extensor digitorum longus; NCC, nerve calcium concentration; NCX, sodium calcium exchanger; NFC, nerve fiber count; PMCA, plasma membrane calcium ATPase; RCR, relative calcium ratio; RFU, relative fluorescent units; TMF, tetanic muscle force Key words: calcitonin; nerve repair; nerve regeneration; osmotic pump; peripheral nerve injury Correspondence to: J.-G. Yan; e-mail [email protected] C 2014 Wiley Periodicals, Inc. V
Published online 9 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.24281
Calcitonin Pump and Nerve Repair
or that directly reducing calcium influx or accelerating calcium absorption can improve nerve regeneration and functional recovery. Therefore, the purpose of this study was to test the effect of an implantable mini-osmotic pump to deliver calcitonin to the transected and repaired nerve. METHODS
For the care and use of laboratory animals, all protocols of this study received approval from the Biomedical Resource Center and the institutional animal care and use committee at the Medical College of Wisconsin. Sixteen healthy 3-month-old Sprague-Dawley rats, weighing between 250 and 300 g, were divided into 2 groups of 8 each, including: (1) a control group (n 5 8), with transection of sciatic nerve at the mid-point and end-to-end repair only; and (2) a calcitonin group (n 5 8), with transection injury and end-toend repair and implantation of a calcitonin pump.
Animal Groups.
Surgery. The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/kg body weight) and prepped for aseptic surgery. A 2-cm incision of the dorsal gluteal area was made on each side, and both sciatic nerves were exposed and dissected free from underlying tissue. The left nerve served as the sham control, and the skin incision was sutured without any further procedures. The right sciatic nerve was transected at the mid-point with a sterile stainless-steel #10 scalpel blade. The transected nerve was allowed to set for 1 hour to induce realistic environmental factors and was then sutured back together. After the procedures, the sciatic nerve was repaired directly by an end-to-end coaptation using 10-0 nylon with 4 sutures. Implanted Mini-Osmotic Pump. An implantable mini-osmotic pump (3 cm in length and 0.7 cm in diameter with a weight of 1.1 g; Alzet; Durect Corp., Cupertino California) with an attached catheter was routed along the injured sciatic nerve (Fig. 1). Ten small needle holes were made in the distal catheter, which continuously and slowly delivered the medications directly to the site of injury over an extended period of time (Fig. 1A). The mini-osmotic pump was implanted subcutaneously dorsally between the scapulae (Fig. 1B), and the catheter was fixed to tissue adjacent to the MUSCLE & NERVE
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FIGURE 1. (A) The Alzet osmotic pump body is 3 cm in length and 0.7 cm in diameter with a reservoir volume of 200 ml (shown with the attached 7-inch catheter). It was implanted subcutaneously between the scapulae of the rat. (B) Implanted osmotic pump (white oval shape). The black line represents the attached flexible catheter. A silicon Elizabethan collar prevented self-mutilation of the deinnervated foot (not shown). The strip represents the transected and repaired sciatic nerve.
injured sciatic nerve by 6/0 nylon. The outer membrane of the pump body is a cellulose ester blend, and the drug reservoir is a thermoplastic hydrocarbon elastomer. The pump has a reservoir volume of 200 ll and is rated to deliver 0.15 ll per hour for 42 days. The pump guaranteed the medications flowed at a constant and gradual rate. The pump was filled with 200 ll of 1.25-lg/ml sterile calcitonin solution (Novartis, East Hanover, New Jersey), which was composed of sterile distilled water that was re-sterilized by passage through a 0.2-lm filter. The concentration of this compound was determined to be approximately the same dose as that given to humans. After 12 weeks of survival time, studies were evaluated and compared between the 2 groups. Electrophysiological studies included compound muscle action potential (CMAP) and tetanic muscle force (TMF), and histological studies included nerve calcium concentration (NCC), relative calcium absorptive ratio (RCR), number of calcified spots (CS), and nerve fiber count (NFC). To avoid animal individual bias, the recovery rate was used for comparison, which was determined by the value on the experimental side (right) divided by the value on the sham control side (left) 3 100%. The RCR was calculated by the average calcium concentration of the treatment (right) divided by the average calcium concentration of contralateral sham control (left), and subtracted from 100%.
Evaluation.
Electrophysiological analysis was performed on both the left (contralateral sham control) and right (treatment) sciatic nerves before tissue harvesting. To measure CMAP, an electromyography system (Sapphire Premier; TECA/Vickers, Pleasantville, New York) was used. The stimulating electrodes were bipolar Teflon-coated stainless-steel electrodes separated by 1 mm. The stimulation sites were on the sciatic nerve proximal to the bifurcation point. At each Compound Muscle Action Potential.
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stimulation site, the cathode was distal, and the anode was proximal. The ground electrode was a Teflon-coated stainless-steel needle inserted into the tail. CMAPs were recorded by 2 monopolar stainless-steel needles inserted 3 mm apart in the belly of the extensor digitorum longus muscle (EDL). The stimulus was a square electric pulse with a voltage of 19 mV, duration of 0.01 ms, and frequency of 1 HZ. During these procedures, the room temperature was controlled at 25 C, and a heating pad maintained rat body temperature at 37 C, as measured by a rectal temperature probe.6 Tetanic Muscle Force Test. After the electrophysiological analysis, tetanic force (contractility) of the EDL muscle was measured. The tendon and belly of the EDL were exposed by incision along the anterior median line of the leg and foot. The EDL tendon was identified and gently retracted to see the digital extension and the muscle belly excursion. A tetanic force machine (Type 386; Stemtech, Inc., Akron, Ohio; and Model DI-205; DATAQ, Akron, Ohio) was used in this study. The bipolar stimulator needles were inserted into the EDL muscle belly center, and the hook of the muscle force transducer was hooked to the EDL tendon. Electrical stimulations to the muscle were delivered at 60 HZ with 60-ms delay and at 52 V with 0.8-ms delay. Isometric muscle contractile force was measured in situ to determine TMF.7 Measurement of Calcium Intensity. Calcium Relative Fluorescent Units. After the CMAP and TMF tests, a 4-mm segment of the sciatic nerve from the transected site was harvested (Fig. 2). Calcium fluorescence of the nerve fibers was measured using our published method.6 The nerves were placed in 1.5-ml Eppendorf tubes with the staining solution, which contained 7 lg of calcium green-1 acetoxymethyl (AM) ester (Molecular Probes/Invitrogen, Carlsbad, California) dissolved in 7 ll of 20% Pluronic F-127/dimethylsulfoxide (Molecular Probes/ MUSCLE & NERVE
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FIGURE 2. Calcium green-1 AM ester fluorescent-stained distal segment of the transected and repaired sciatic nerve after 12 weeks. (A) Normal control nerve shows homogeneous slightly bright calcium staining throughout the myelin sheath. (B) A transected and repaired nerve with a calcitonin osmotic pump. Calcium is shown as homogeneous and slightly bright green and is similar to normal control. (C) Transected and repaired nerve without a calcitonin pump. This nerve has the highest intensity of fluorescence throughout the nerve trunk, which correlates with the largest accumulation of calcium and poorest function. Bar 5 100 lm.
Invitrogen) and further diluted in 1 ml of Dulbecco modified Eagle medium without phenol red. Using 25-gauge needles, the nerves were separated carefully such that small fiber bundles were visible. They were viewed under fluorescent conditions with a Zeiss microscope (Axio Scope 40; Zeiss Microimaging, Thornwood, New York). Images were composed of a general representative portion of the nerve bundle as well as the background, acquired with a camera and software (AxioCam MRc and AxioVision Imaging; Zeiss MicroImaging), and analyzed with MetaVue software (Molecular Devices, Downington, Pennsylvania). Under these conditions, calcium appears bright green within the nerve fiber bundles with a relatively dark background. Pictures were taken from each nerve sample using the same parameters, and all functional settings were kept at the same values throughout the analysis (brightness 5 20.5, contrast 5 1.0, and gain 5 1.0). All pictures were taken at 2003 magnification. The calcium fluorescence level was calculated and expressed in relative fluorescent units (RFU) using the MetaVue software, which is designed to measure brightness intensity. The background value was subtracted from the nerve segment value to allow for any fluctuations in overall fluorescence. Nerve Calcium Concentration. It has been documented that human and all other mammalian neural systems have homeostatic mechanisms to adjust intracellular (1 3 1027 M) and extracellular (1 3 1023 M) concentrations of calcium ions.5 We used this fact to calculate the calcium concentrations in the tested nerves. Because we cannot obtain accurate RFU values from inside the cell (axoplasm) due to the myelin membrane (which has a high level of calcium), we measured the extracellular matrix. This is the area between normal nerve fibers, and its mean value is 14.16 6 1.34 RFU. This number is equal to the calcium concentration in the extracellular environment, which has been Calcitonin Pump and Nerve Repair
found to be 1 3 1023 M. One RFU would be equal to a calcium concentration of 7.14 3 1025 M. Using basic algebra, we calculated the calcium concentrations of the various nerve segments from earlier studies.6–8 Sciatic nerve segments 4 mm in length were also harvested 3 mm distal to the transection site. Slides were prepared as follows. The specimens were immersed directly into a 2.5% glutaraldehyde/phosphate-buffered saline. They were then processed routinely and embedded in a mixture consisting of 76.0 g Medcast, 18.0 g Araldite 502, 39.0 g dodecenyl succinic anhydride, 61.0 g nadic methyl anhydride, and 2.0–3.5% 2,4,6tri (dimethyl aminomethyl) phenol-30 by volume (Ted Pella, Inc., Redding, California). Semi-thin 0.5-lm transverse sections were cut and stained with toluidine blue for nerve fiber counting and light microscopy. Histology Study.
Calcified Spots. CS were degenerated myelinated nerve fibers occupied by calcium accumulation spots. There were no dark blue spots in the normal sections (Fig. 3A). There were a number of dark blue, solid ball–like structures in the semi-thin 0.5-lm crosssections stained with toluidine blue (Fig. 3B and C). These spots were also validated by calcium fluorescence stain, shown as bright green spots in Figure 2. More CS meant more marked calcium accumulation. Analysis. Mean 6 standard deviation (SD) results were calculated for normally distributed data. For comparison among the 3 groups (control group, calcitonin pump group, and contralateral sham control group) an analysis of variance test was used. For comparison between 2 groups (control group vs. calcitonin pump group), a 2-sample t-test by 2 sides was used. The correlation coefficient (r) between nerve CMAP recovery rate and RCR was determined by Pearson correlation test.
Statistical
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FIGURE 3. Semi-thin epoxy cross-sections (0.5 lm) of the transected and repaired sciatic nerves stained with toluidine blue after 12 weeks. (A) Normal control nerve. (B) Transected and repaired nerve with calcitonin pump. Thin arrows pointing to hollow circles indicate healthy axons. Arrowhead indicates the central artery of the sciatic nerve. (C) Transected and repaired nerve without a calcitonin pump. Arrowhead indicates the central nerve artery, which has a narrow lumen and very thick arterial wall with smooth muscle hypertrophy, as compared with (B). Thin arrows show disrupted myelinated fibers. The dark circular spots indicate higher calcium levels. Numbers of these calcium accumulation spots in (C) were higher than in (B). Bar 5 100 lm.
RESULTS
The RFU value is the calculated calcium intensity of the entire nerve segment (Table 1). The results are as follows (Mean 6 SD): sham control group, 24.25 6 1.37 RFU; control group, 40.59 6 4.92 RFU; and calcitonin pump group, 32.3 6 2.38 RFU. There were statistically significant differences within the 2 groups (P < 0.001). Relative Fluorescent Units.
Compound Muscle Action Potential. The results of the electrophysiological studies are summarized in Table 2. The recovery rates (injured right side divided by normal left side) of CMAPs were as follows: (1) control group, 66.9 6 8.8%; and (2) calcitonin pump group, 83.6 6 5.4%. The higher value in the calcitonin pump group represented a statistically significant difference (P < 0.001).
The recovery rates of tetanic muscle force (TMF) were as follows: (1) control group, 56.0 6 4.4%; and (2) calcitonin pump group, 82.3 6 4.6% (Table 2). The higher value in the calcitonin pump group represented a statistically significant difference (P < 0.001). Tetanic Muscle Force Test.
The results of the histological study are summarized in Table 1. The average myelinated nerve fiber counts for the injured right side (Table 1) were: (1) control group, 3264 6 305; and (2) calcitonin group (transaction repair with calcitonin pump), 4011 6 428. The recovery rates for average myelinated nerve fiber count were: (1) control group, 66.7 6 1.9%; and (2) calcitonin pump group, 83.2 6 3.9%. The value in the calcitonin pump group was greater than that of the control group in both cases, and the differences were statistically significant (NFC: P < 0.001; NFC recovery rate: P < 0.001). Nerve Fiber Counting.
Calcified Spot Numbers. The CS counting results were: (1) control group, 122 6 51.0; and (2) calcito232
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nin pump group, 31.0 6 17.0. The CS number of the control group was significantly greater than that of the calcitonin pump group, and the difference was statistically significant (P < 0.005). Relative Calcium Absorptive Ratio. The RCR was determined by the average calcium concentration of the treatment (right) divided by the average calcium concentration of sham control (left), and subtracted from 100%. The more RCR, the more calcium absorption, with less calcium on the experimental side. The RCRs were: (1) control group, 58.8 6 3.4%; and (2) calcitonin pump group, 73.9 6 3.9%. The RCR of the calcitonin pump group was significantly greater than that of the control group, and the difference was statistically significant (P < 0.0001). Correlation of CMAP Recovery Rate with Relative Calcium Absorptive Ratio. The correlation coefficient (r) between the nerve CMAP recovery rate and RCR in the calcitonin pump group was 0.80 (P < 0.001). DISCUSSION
Many studies, both in vitro and in vivo, support the association between calcium influx and damage to neural tissues.9–14 High levels of calcium are a direct result of injury to the cell membrane due to Table 1. Results of histological studies. Group NFC RFU RCR (%) CS number
Control
Calcitonin pump
3264 6 305 40.59 6 4.92 58.8 6 3.4 122 6 51.0
4011 6 428† 32.30 6 2.38† 73.9 6 3.9† 31.0 6 17.0*
NFC, nerve fiber count; RFU, relative fluorescent units; RCR, relative calcium ratio (nerve calcium concentration of normal control / group); CS, calcified spot. *Statistically significant difference (P < 0.005). †
Highly statistically significant difference (P < 0.001).
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Table 2. Results of electrophysiological studies. Group CMAP (mV) L R R/L (%) TMF (g) L R R/L (%)
Control
Calcitonin pump
17.7 6 1.5 11.8 6 2.1 66.9 6 8.8
18.5 6 3.4 15.4 6 2.6 83.6 6 5.4*
131.8 6 18.4 74.0 6 13.1 56.0 6 4.4
219 6 35.6 187.9 6 38.7 82.3 6 4.6*
CMAP, compound muscle action potential; TMF, tetanic muscle force; L, left side (normal side); R, right side (experimental side); R/L (%), recovery rate. *Highly statistically significant difference (P < 0.001).
mechanical insults15–17 or ischemic events.18 Very little research has been conducted on injured and recovering peripheral nerves with regard to calcium concentrations. Our previous study showed that, after nerve crush injury, functional recovery was correlated statistically with calcium absorption.6,19 Also, our recent work showed that accelerated calcium absorption can greatly improve nerve regeneration.5,20 Neurotoxins, as well as excitatory amino acids, induce nerve degeneration in combination with high levels of calcium.11 High calcium concentration can also cause overstimulation of enzymes, such as phospholipases, plasmalogenase, calpains and other proteases, protein kinases, guanylate cyclase, nitric oxide synthase, calcineurins, and endonucleases. Overstimulation of these enzymes can lead to an overproduction of toxic reaction products, such as free radicals, lethal alterations in cytoskeletal organization, or activation of genetic signals that lead to cell death.9 Although advanced microsurgical techniques are available, we lack understanding of the nerve injury cascade process and the cellular and molecular mechanism of nerve degeneration and regeneration. This study once again confirmed our previous study findings that the degree of nerve injury is very strongly correlated with calcium accumulation.6 A recent study by our group showed that excessive calcium is detrimental to Schwann cell proliferation and growth.8 We found that NCC and CS provided objective evidence for calcium accumulation and its harmful effects. Calcium absorption medications dramatically decreased NCC and CS, which can improve nerve regeneration. In Figure 3 the arrowhead indicates the central nerve artery, which has a narrow lumen and very thick artery wall due to smooth muscle hypertrophy. This is because excessive calcium induced consistent smooth muscle contraction as well-known rigor status, which over time causes vascular constriction and reduced nerve blood supply. Based on these findings, a new Calcitonin Pump and Nerve Repair
concept has been formulated that excessive calcium induces an injury cascade, which is a very severe form of secondary injury. This secondary injury cascade further worsens the primary injury and impedes nerve regeneration. A novel approach with potential clinical translation based on the above findings was our attempt to lower calcium levels after injury by delivering calcium-absorbing medications. To achieve a continuous and gradual mode of delivery, a mini-osmotic pump was implanted to deliver medication at a constant 0.15 ll/h. Results with the micro-osmotic pumps with the calcium-absorbing medication calcitonin were far superior to those of the sham controls. It is known that blood–nerve barrier (BNB) permeability depends on molecular size. Macromolecular albumin and IgG have difficulty in passing the BNB; another special receptor mechanism allows macromolecular nerve growth factor to easily pass the BNB.21 Calcitonin (also known as thyrocalcitonin) is a 32-amino-acid linear polypeptide hormone that is produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid and in many other animals in the ultimobranchial body. Although it is a macromolecular polypeptide, there is a special receptor for calcitonin on the peripheral nerve. Gene expression of the calcitonin receptor and binding site is increased at transected and chronically constricted peripheral nerve tissue areas.22,23 It acts to reduce blood calcium (Ca21), opposing the effects of parathyroid hormone. Longterm studies using intranasal calcitonin for relief of phantom limb pain and injured nerve pain have been reported.24 Calcitonin’s local efficacy was confirmed in the above study, which was effective on peripheral nerves. In our study, the precise local action mechanism of calcitonin for calcium absorption on nerve is unclear. It may directly affect the nerve banded with its receptor on the injured and repaired nerve. Alternately, it may have affected the adjacent femur and decreased local Ca21 concentration of the surrounding tissue of the sciatic nerve, thereby extracting Ca21 from the injured nerve; however, this is implausible, because the dose used in the pump was too low to distribute over the body. Calcitonin has short absorption and elimination half-lives of 10–15 minutes and 50–80 minutes, respectively; however, using an osmotic pump allows for gradual and prolonged release.25 We conclude that: 1. Nerve regeneration and function recovery after nerve repair correlate strongly with calcium absorption. 2. Calcitonin has a direct effect on the injured and repaired peripheral nerve. MUSCLE & NERVE
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3. The calcitonin-filled mini-osmotic pump offers greatly improved nerve regeneration and functional recovery due to the calcitonin directly accelerating calcium absorption from the repaired nerve, which can potentially be translated into clinical applications. REFERENCES 1. Spitzer NC, Gu X, Olson E. Action potentials, calcium transients and the control of differentiation of excitable cells. Curr Opin Neurobiol 1994;4:70–77. 2. Yuste R, Katz LC. Control of postsynaptic Ca21 influx in developing neocortex by excitatory and inhibitory neurotransmitters. Neuron 1991;6:333–344. 3. Blaustein MP. Calcium transport and buffering in neurons. Trends Neurosci 1988;11:438–443. 4. Stys PK, Ransom BR, Waxman SG, Davis PK. Role of extracellular calcium in anoxic injury of mammalian central white matter. Proc Natl Acad Sci USA 1990;87:4212–4216. 5. Kiedrowski LG, Brooker G, Costa E, Wroblewski JT. Glutamate impairs neuronal calcium extrusion while reducing sodium gradient. Neuron 1994;12:295–300. 6. Yan JG, Matloub HS, Yan Y, Agresti M, Zhang LL, Jaradeh SS. The correlation between calcium absorption and electrophysiological recovery in crushed rat peripheral nerves. Microsurgery 2010;30:138– 145. 7. Yan YH, Yan JG, Sanger JR, Zhang LL, Riley DA, Matloub HS. Nerve repair at different angles of attachment: experiment in rats. J Reconstr Microsurg 2002;18:703–708. 8. Yan JG, Agresti M, Zhang LL, Matloub HS, Sanger JR. Negative effect of high calcium levels on Schwann cell survival. Neurophysiology 2012;44:274–278. 9. LoPachin RM, Lehning EJ. Mechanism of calcium entry during axon injury and degeneration. Toxicol Appl Pharmacol 1997;143:233–244. 10. Usachev YM, DeMarco SJ, Campbell C, Strehler EE, Thayer SA. Bradykinin and ATP accelerate Ca(21) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(21) pump isoform 4. Neuron 2002;33:113–122. 11. Jancso G, Karcsu S, Kiraly E, Szebeni A, Toth L, Bacsy E, et al. Neurotoxin induced nerve cell degeneration: possible involvement of calcium. Brain Res 1984;295:211–216.
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12. Goldberg MP, Giffard RG, Kurth MC, Choi DW. Role of extracellular calcium and magnesium in ischemic neuronal injury in vitro. Neurology 1989;39(suppl):217. 13. Dietz RM, Weiss JH, Shuttleworth CW. Contributions of Ca21 and Zn21 to spreading depression-like events and neuronal injury. J Neurochem 2009;109(suppl):145–152. 14. Ma YY, Li KY, Wang JJ, Huang YL, Huang Y, Sun FY. Vascular endothelial growth factor acutely reduces calcium influx via inhibition of the Ca21 channels in rat hippocampal neurons. J Neurosci Res 2009;87:393–402. 15. Lucas JH, Gross GW, Emery DG, Gardner CR. Neuronal survival or death after dendrite transection close to the perikaryon: correlation with electrophysiologic, morphologic, and ultrastructural changes. Cent Nerv Syst Trauma 1985;2:231–255. 16. Shi RY, Lucas JH, Wolf A, Gross GW. Calcium antagonists fail to protect mammalian spinal neurons after physical injury. J Neurotrauma 1989;6:261–278. 17. Strautman AF, Cork RJ, Robinson KR. The distribution of free calcium in transected spinal axons and its modulation by applied electrical fields. J Neurosci 1990;10:3564–3575. 18. Rehncrona S, Mela L, Siesjo BK. Recovery of brain mitochondrial function in the rat after complete and incomplete cerebral ischemia. Stroke 1979;10:437–446. 19. Yan JG, Zhang LL, Agresti M, Logiudice J, Yan YH, Wang Z, Sanger JR, Matloub HS. The effect of calcium modulating agents on peripheral nerve recovery after crush. J Neurosci Methods 2013;15;217:54– 62. 20. Mattson MP, Guthrie PB, Kater SB. A role for Na1-dependent Ca21 extrusion in protection against neuronal excitotoxicity. FASEB J 1989;3:2519–2526. 21. Poduslo JF, Curran GL, Berg CT. Macromolecular permeability across the blood–nerve and blood–brain barriers. Proc Natl Acad Sci USA 1994;91:5705–5709. 22. Sauer SK, Bove GM, Averbeck B, Reeh PW. Rat peripheral nerve components release calcitonin gene-related peptide and prostaglandin E2 in response to noxious stimuli: evidence that nervi nervorum are nociceptors. Neuroscience 1999;92:319–325. 23. Ito A, Takeda M. Anti-hyperalgesic effects of calcitonin on neuropathic pain interacting with its peripheral receptors. Mol Pain 2012; 8:42. 24. Hirsch S, Corradini L, Just S, Arndt K, Doods H. The CGRP receptor antagonist BIBN4096BS peripherally alleviates inflammatory pain in rats. Pain 2013;154:700–707. 25. Murdoch FR, Maker GL, Nitsos I, Polglase GR, Musk GC. Intraperitoneal medetomidine: a novel analgesic strategy for postoperative pain management in pregnant sheep. Lab Anim 2013;47:66–70.
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Calcium ions play a crucial role in normal neuronal function.1,2 Intraneural scarring is ubiquitous after peripheral nerve injury, which damages numerous membrane transporters, including plasma membrane calcium ATPase (PMCA) and sodium calcium exchanger (NCX). PMCA and NCX normally act as the main mechanisms of calcium extrusion to maintain a much lower intracellular concentration of calcium relative to the extracellular concentration. The large calcium concentration gradient is necessary for a wide variety of cell signaling and transduction events. However, when PMCA and NCX are damaged, calcium influx via diffusion then overwhelms the calcium extrusion mechanisms.3,4 The resultant rise in intracellular calcium can disrupt normal cell signaling and transductions and can activate secondary Ca21-dependent cascades, such as apoptosis, resulting in Schwann cell death.5 There have not been any studies attempting to validate the theory that excessive calcium impedes nerve regeneration Abbreviations: AM, acetoxy methyl; BNB, blood–nerve barrier; CMAP, compound muscle action potential; CS, calcified spot; EDL, extensor digitorum longus; NCC, nerve calcium concentration; NCX, sodium calcium exchanger; NFC, nerve fiber count; PMCA, plasma membrane calcium ATPase; RCR, relative calcium ratio; RFU, relative fluorescent units; TMF, tetanic muscle force Key words: calcitonin; nerve repair; nerve regeneration; osmotic pump; peripheral nerve injury Correspondence to: J.-G. Yan; e-mail [email protected] C 2014 Wiley Periodicals, Inc. V
Published online 9 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.24281
Calcitonin Pump and Nerve Repair
or that directly reducing calcium influx or accelerating calcium absorption can improve nerve regeneration and functional recovery. Therefore, the purpose of this study was to test the effect of an implantable mini-osmotic pump to deliver calcitonin to the transected and repaired nerve. METHODS
For the care and use of laboratory animals, all protocols of this study received approval from the Biomedical Resource Center and the institutional animal care and use committee at the Medical College of Wisconsin. Sixteen healthy 3-month-old Sprague-Dawley rats, weighing between 250 and 300 g, were divided into 2 groups of 8 each, including: (1) a control group (n 5 8), with transection of sciatic nerve at the mid-point and end-to-end repair only; and (2) a calcitonin group (n 5 8), with transection injury and end-toend repair and implantation of a calcitonin pump.
Animal Groups.
Surgery. The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/kg body weight) and prepped for aseptic surgery. A 2-cm incision of the dorsal gluteal area was made on each side, and both sciatic nerves were exposed and dissected free from underlying tissue. The left nerve served as the sham control, and the skin incision was sutured without any further procedures. The right sciatic nerve was transected at the mid-point with a sterile stainless-steel #10 scalpel blade. The transected nerve was allowed to set for 1 hour to induce realistic environmental factors and was then sutured back together. After the procedures, the sciatic nerve was repaired directly by an end-to-end coaptation using 10-0 nylon with 4 sutures. Implanted Mini-Osmotic Pump. An implantable mini-osmotic pump (3 cm in length and 0.7 cm in diameter with a weight of 1.1 g; Alzet; Durect Corp., Cupertino California) with an attached catheter was routed along the injured sciatic nerve (Fig. 1). Ten small needle holes were made in the distal catheter, which continuously and slowly delivered the medications directly to the site of injury over an extended period of time (Fig. 1A). The mini-osmotic pump was implanted subcutaneously dorsally between the scapulae (Fig. 1B), and the catheter was fixed to tissue adjacent to the MUSCLE & NERVE
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FIGURE 1. (A) The Alzet osmotic pump body is 3 cm in length and 0.7 cm in diameter with a reservoir volume of 200 ml (shown with the attached 7-inch catheter). It was implanted subcutaneously between the scapulae of the rat. (B) Implanted osmotic pump (white oval shape). The black line represents the attached flexible catheter. A silicon Elizabethan collar prevented self-mutilation of the deinnervated foot (not shown). The strip represents the transected and repaired sciatic nerve.
injured sciatic nerve by 6/0 nylon. The outer membrane of the pump body is a cellulose ester blend, and the drug reservoir is a thermoplastic hydrocarbon elastomer. The pump has a reservoir volume of 200 ll and is rated to deliver 0.15 ll per hour for 42 days. The pump guaranteed the medications flowed at a constant and gradual rate. The pump was filled with 200 ll of 1.25-lg/ml sterile calcitonin solution (Novartis, East Hanover, New Jersey), which was composed of sterile distilled water that was re-sterilized by passage through a 0.2-lm filter. The concentration of this compound was determined to be approximately the same dose as that given to humans. After 12 weeks of survival time, studies were evaluated and compared between the 2 groups. Electrophysiological studies included compound muscle action potential (CMAP) and tetanic muscle force (TMF), and histological studies included nerve calcium concentration (NCC), relative calcium absorptive ratio (RCR), number of calcified spots (CS), and nerve fiber count (NFC). To avoid animal individual bias, the recovery rate was used for comparison, which was determined by the value on the experimental side (right) divided by the value on the sham control side (left) 3 100%. The RCR was calculated by the average calcium concentration of the treatment (right) divided by the average calcium concentration of contralateral sham control (left), and subtracted from 100%.
Evaluation.
Electrophysiological analysis was performed on both the left (contralateral sham control) and right (treatment) sciatic nerves before tissue harvesting. To measure CMAP, an electromyography system (Sapphire Premier; TECA/Vickers, Pleasantville, New York) was used. The stimulating electrodes were bipolar Teflon-coated stainless-steel electrodes separated by 1 mm. The stimulation sites were on the sciatic nerve proximal to the bifurcation point. At each Compound Muscle Action Potential.
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stimulation site, the cathode was distal, and the anode was proximal. The ground electrode was a Teflon-coated stainless-steel needle inserted into the tail. CMAPs were recorded by 2 monopolar stainless-steel needles inserted 3 mm apart in the belly of the extensor digitorum longus muscle (EDL). The stimulus was a square electric pulse with a voltage of 19 mV, duration of 0.01 ms, and frequency of 1 HZ. During these procedures, the room temperature was controlled at 25 C, and a heating pad maintained rat body temperature at 37 C, as measured by a rectal temperature probe.6 Tetanic Muscle Force Test. After the electrophysiological analysis, tetanic force (contractility) of the EDL muscle was measured. The tendon and belly of the EDL were exposed by incision along the anterior median line of the leg and foot. The EDL tendon was identified and gently retracted to see the digital extension and the muscle belly excursion. A tetanic force machine (Type 386; Stemtech, Inc., Akron, Ohio; and Model DI-205; DATAQ, Akron, Ohio) was used in this study. The bipolar stimulator needles were inserted into the EDL muscle belly center, and the hook of the muscle force transducer was hooked to the EDL tendon. Electrical stimulations to the muscle were delivered at 60 HZ with 60-ms delay and at 52 V with 0.8-ms delay. Isometric muscle contractile force was measured in situ to determine TMF.7 Measurement of Calcium Intensity. Calcium Relative Fluorescent Units. After the CMAP and TMF tests, a 4-mm segment of the sciatic nerve from the transected site was harvested (Fig. 2). Calcium fluorescence of the nerve fibers was measured using our published method.6 The nerves were placed in 1.5-ml Eppendorf tubes with the staining solution, which contained 7 lg of calcium green-1 acetoxymethyl (AM) ester (Molecular Probes/Invitrogen, Carlsbad, California) dissolved in 7 ll of 20% Pluronic F-127/dimethylsulfoxide (Molecular Probes/ MUSCLE & NERVE
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FIGURE 2. Calcium green-1 AM ester fluorescent-stained distal segment of the transected and repaired sciatic nerve after 12 weeks. (A) Normal control nerve shows homogeneous slightly bright calcium staining throughout the myelin sheath. (B) A transected and repaired nerve with a calcitonin osmotic pump. Calcium is shown as homogeneous and slightly bright green and is similar to normal control. (C) Transected and repaired nerve without a calcitonin pump. This nerve has the highest intensity of fluorescence throughout the nerve trunk, which correlates with the largest accumulation of calcium and poorest function. Bar 5 100 lm.
Invitrogen) and further diluted in 1 ml of Dulbecco modified Eagle medium without phenol red. Using 25-gauge needles, the nerves were separated carefully such that small fiber bundles were visible. They were viewed under fluorescent conditions with a Zeiss microscope (Axio Scope 40; Zeiss Microimaging, Thornwood, New York). Images were composed of a general representative portion of the nerve bundle as well as the background, acquired with a camera and software (AxioCam MRc and AxioVision Imaging; Zeiss MicroImaging), and analyzed with MetaVue software (Molecular Devices, Downington, Pennsylvania). Under these conditions, calcium appears bright green within the nerve fiber bundles with a relatively dark background. Pictures were taken from each nerve sample using the same parameters, and all functional settings were kept at the same values throughout the analysis (brightness 5 20.5, contrast 5 1.0, and gain 5 1.0). All pictures were taken at 2003 magnification. The calcium fluorescence level was calculated and expressed in relative fluorescent units (RFU) using the MetaVue software, which is designed to measure brightness intensity. The background value was subtracted from the nerve segment value to allow for any fluctuations in overall fluorescence. Nerve Calcium Concentration. It has been documented that human and all other mammalian neural systems have homeostatic mechanisms to adjust intracellular (1 3 1027 M) and extracellular (1 3 1023 M) concentrations of calcium ions.5 We used this fact to calculate the calcium concentrations in the tested nerves. Because we cannot obtain accurate RFU values from inside the cell (axoplasm) due to the myelin membrane (which has a high level of calcium), we measured the extracellular matrix. This is the area between normal nerve fibers, and its mean value is 14.16 6 1.34 RFU. This number is equal to the calcium concentration in the extracellular environment, which has been Calcitonin Pump and Nerve Repair
found to be 1 3 1023 M. One RFU would be equal to a calcium concentration of 7.14 3 1025 M. Using basic algebra, we calculated the calcium concentrations of the various nerve segments from earlier studies.6–8 Sciatic nerve segments 4 mm in length were also harvested 3 mm distal to the transection site. Slides were prepared as follows. The specimens were immersed directly into a 2.5% glutaraldehyde/phosphate-buffered saline. They were then processed routinely and embedded in a mixture consisting of 76.0 g Medcast, 18.0 g Araldite 502, 39.0 g dodecenyl succinic anhydride, 61.0 g nadic methyl anhydride, and 2.0–3.5% 2,4,6tri (dimethyl aminomethyl) phenol-30 by volume (Ted Pella, Inc., Redding, California). Semi-thin 0.5-lm transverse sections were cut and stained with toluidine blue for nerve fiber counting and light microscopy. Histology Study.
Calcified Spots. CS were degenerated myelinated nerve fibers occupied by calcium accumulation spots. There were no dark blue spots in the normal sections (Fig. 3A). There were a number of dark blue, solid ball–like structures in the semi-thin 0.5-lm crosssections stained with toluidine blue (Fig. 3B and C). These spots were also validated by calcium fluorescence stain, shown as bright green spots in Figure 2. More CS meant more marked calcium accumulation. Analysis. Mean 6 standard deviation (SD) results were calculated for normally distributed data. For comparison among the 3 groups (control group, calcitonin pump group, and contralateral sham control group) an analysis of variance test was used. For comparison between 2 groups (control group vs. calcitonin pump group), a 2-sample t-test by 2 sides was used. The correlation coefficient (r) between nerve CMAP recovery rate and RCR was determined by Pearson correlation test.
Statistical
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FIGURE 3. Semi-thin epoxy cross-sections (0.5 lm) of the transected and repaired sciatic nerves stained with toluidine blue after 12 weeks. (A) Normal control nerve. (B) Transected and repaired nerve with calcitonin pump. Thin arrows pointing to hollow circles indicate healthy axons. Arrowhead indicates the central artery of the sciatic nerve. (C) Transected and repaired nerve without a calcitonin pump. Arrowhead indicates the central nerve artery, which has a narrow lumen and very thick arterial wall with smooth muscle hypertrophy, as compared with (B). Thin arrows show disrupted myelinated fibers. The dark circular spots indicate higher calcium levels. Numbers of these calcium accumulation spots in (C) were higher than in (B). Bar 5 100 lm.
RESULTS
The RFU value is the calculated calcium intensity of the entire nerve segment (Table 1). The results are as follows (Mean 6 SD): sham control group, 24.25 6 1.37 RFU; control group, 40.59 6 4.92 RFU; and calcitonin pump group, 32.3 6 2.38 RFU. There were statistically significant differences within the 2 groups (P < 0.001). Relative Fluorescent Units.
Compound Muscle Action Potential. The results of the electrophysiological studies are summarized in Table 2. The recovery rates (injured right side divided by normal left side) of CMAPs were as follows: (1) control group, 66.9 6 8.8%; and (2) calcitonin pump group, 83.6 6 5.4%. The higher value in the calcitonin pump group represented a statistically significant difference (P < 0.001).
The recovery rates of tetanic muscle force (TMF) were as follows: (1) control group, 56.0 6 4.4%; and (2) calcitonin pump group, 82.3 6 4.6% (Table 2). The higher value in the calcitonin pump group represented a statistically significant difference (P < 0.001). Tetanic Muscle Force Test.
The results of the histological study are summarized in Table 1. The average myelinated nerve fiber counts for the injured right side (Table 1) were: (1) control group, 3264 6 305; and (2) calcitonin group (transaction repair with calcitonin pump), 4011 6 428. The recovery rates for average myelinated nerve fiber count were: (1) control group, 66.7 6 1.9%; and (2) calcitonin pump group, 83.2 6 3.9%. The value in the calcitonin pump group was greater than that of the control group in both cases, and the differences were statistically significant (NFC: P < 0.001; NFC recovery rate: P < 0.001). Nerve Fiber Counting.
Calcified Spot Numbers. The CS counting results were: (1) control group, 122 6 51.0; and (2) calcito232
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nin pump group, 31.0 6 17.0. The CS number of the control group was significantly greater than that of the calcitonin pump group, and the difference was statistically significant (P < 0.005). Relative Calcium Absorptive Ratio. The RCR was determined by the average calcium concentration of the treatment (right) divided by the average calcium concentration of sham control (left), and subtracted from 100%. The more RCR, the more calcium absorption, with less calcium on the experimental side. The RCRs were: (1) control group, 58.8 6 3.4%; and (2) calcitonin pump group, 73.9 6 3.9%. The RCR of the calcitonin pump group was significantly greater than that of the control group, and the difference was statistically significant (P < 0.0001). Correlation of CMAP Recovery Rate with Relative Calcium Absorptive Ratio. The correlation coefficient (r) between the nerve CMAP recovery rate and RCR in the calcitonin pump group was 0.80 (P < 0.001). DISCUSSION
Many studies, both in vitro and in vivo, support the association between calcium influx and damage to neural tissues.9–14 High levels of calcium are a direct result of injury to the cell membrane due to Table 1. Results of histological studies. Group NFC RFU RCR (%) CS number
Control
Calcitonin pump
3264 6 305 40.59 6 4.92 58.8 6 3.4 122 6 51.0
4011 6 428† 32.30 6 2.38† 73.9 6 3.9† 31.0 6 17.0*
NFC, nerve fiber count; RFU, relative fluorescent units; RCR, relative calcium ratio (nerve calcium concentration of normal control / group); CS, calcified spot. *Statistically significant difference (P < 0.005). †
Highly statistically significant difference (P < 0.001).
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Table 2. Results of electrophysiological studies. Group CMAP (mV) L R R/L (%) TMF (g) L R R/L (%)
Control
Calcitonin pump
17.7 6 1.5 11.8 6 2.1 66.9 6 8.8
18.5 6 3.4 15.4 6 2.6 83.6 6 5.4*
131.8 6 18.4 74.0 6 13.1 56.0 6 4.4
219 6 35.6 187.9 6 38.7 82.3 6 4.6*
CMAP, compound muscle action potential; TMF, tetanic muscle force; L, left side (normal side); R, right side (experimental side); R/L (%), recovery rate. *Highly statistically significant difference (P < 0.001).
mechanical insults15–17 or ischemic events.18 Very little research has been conducted on injured and recovering peripheral nerves with regard to calcium concentrations. Our previous study showed that, after nerve crush injury, functional recovery was correlated statistically with calcium absorption.6,19 Also, our recent work showed that accelerated calcium absorption can greatly improve nerve regeneration.5,20 Neurotoxins, as well as excitatory amino acids, induce nerve degeneration in combination with high levels of calcium.11 High calcium concentration can also cause overstimulation of enzymes, such as phospholipases, plasmalogenase, calpains and other proteases, protein kinases, guanylate cyclase, nitric oxide synthase, calcineurins, and endonucleases. Overstimulation of these enzymes can lead to an overproduction of toxic reaction products, such as free radicals, lethal alterations in cytoskeletal organization, or activation of genetic signals that lead to cell death.9 Although advanced microsurgical techniques are available, we lack understanding of the nerve injury cascade process and the cellular and molecular mechanism of nerve degeneration and regeneration. This study once again confirmed our previous study findings that the degree of nerve injury is very strongly correlated with calcium accumulation.6 A recent study by our group showed that excessive calcium is detrimental to Schwann cell proliferation and growth.8 We found that NCC and CS provided objective evidence for calcium accumulation and its harmful effects. Calcium absorption medications dramatically decreased NCC and CS, which can improve nerve regeneration. In Figure 3 the arrowhead indicates the central nerve artery, which has a narrow lumen and very thick artery wall due to smooth muscle hypertrophy. This is because excessive calcium induced consistent smooth muscle contraction as well-known rigor status, which over time causes vascular constriction and reduced nerve blood supply. Based on these findings, a new Calcitonin Pump and Nerve Repair
concept has been formulated that excessive calcium induces an injury cascade, which is a very severe form of secondary injury. This secondary injury cascade further worsens the primary injury and impedes nerve regeneration. A novel approach with potential clinical translation based on the above findings was our attempt to lower calcium levels after injury by delivering calcium-absorbing medications. To achieve a continuous and gradual mode of delivery, a mini-osmotic pump was implanted to deliver medication at a constant 0.15 ll/h. Results with the micro-osmotic pumps with the calcium-absorbing medication calcitonin were far superior to those of the sham controls. It is known that blood–nerve barrier (BNB) permeability depends on molecular size. Macromolecular albumin and IgG have difficulty in passing the BNB; another special receptor mechanism allows macromolecular nerve growth factor to easily pass the BNB.21 Calcitonin (also known as thyrocalcitonin) is a 32-amino-acid linear polypeptide hormone that is produced in humans primarily by the parafollicular cells (also known as C-cells) of the thyroid and in many other animals in the ultimobranchial body. Although it is a macromolecular polypeptide, there is a special receptor for calcitonin on the peripheral nerve. Gene expression of the calcitonin receptor and binding site is increased at transected and chronically constricted peripheral nerve tissue areas.22,23 It acts to reduce blood calcium (Ca21), opposing the effects of parathyroid hormone. Longterm studies using intranasal calcitonin for relief of phantom limb pain and injured nerve pain have been reported.24 Calcitonin’s local efficacy was confirmed in the above study, which was effective on peripheral nerves. In our study, the precise local action mechanism of calcitonin for calcium absorption on nerve is unclear. It may directly affect the nerve banded with its receptor on the injured and repaired nerve. Alternately, it may have affected the adjacent femur and decreased local Ca21 concentration of the surrounding tissue of the sciatic nerve, thereby extracting Ca21 from the injured nerve; however, this is implausible, because the dose used in the pump was too low to distribute over the body. Calcitonin has short absorption and elimination half-lives of 10–15 minutes and 50–80 minutes, respectively; however, using an osmotic pump allows for gradual and prolonged release.25 We conclude that: 1. Nerve regeneration and function recovery after nerve repair correlate strongly with calcium absorption. 2. Calcitonin has a direct effect on the injured and repaired peripheral nerve. MUSCLE & NERVE
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