Biomedical subjects

Daniel A Hunter

Publications and source records attributed to Daniel A Hunter.

35 records · Page 2Linked to original sources

PubMed2004

Effects of motor versus sensory nerve grafts on peripheral nerve regeneration.

Autologous nerve grafting is the current standard of care for nerve injuries resulting in a nerve gap. This treatment requires the use of sensory grafts to reconstruct motor defects, but the consequences of mismatches between graft and native nerve are unknown. Motor pathways have been shown to preferentially support motoneuron regeneration. Functional outcome of motor nerve reconstruction depends on the magnitude, rate, and precision of end organ reinnervation. This study examined the role of pathway type on regeneration across a mixed nerve defect. Thirty-six Lewis rats underwent tibial nerve transection and received isogeneic motor, sensory or mixed nerve grafts. Histomorphometry of the regenerating nerves at 3 weeks demonstrated robust nerve regeneration through both motor and mixed nerve grafts. In contrast, poor nerve regeneration was seen through sensory nerve grafts, with significantly decreased nerve fiber count, percent nerve, and nerve density when compared with mixed and motor groups (P < 0.05). These data suggest that use of motor or mixed nerve grafts, rather than sensory nerve grafts, will optimize regeneration across mixed nerve defects.

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PubMed2004

A novel model for the study of peripheral-nerve regeneration following common nerve injury paradigms.

Recent advances in molecular neurobiology include the development of transgenic mice that express genes encoding fluorescent proteins under neuron-specific promoters (XFP mice). These mice have been used in the field of developmental neurobiology, but use has expanded to include the study of peripheral-nerve axonal regeneration subsequent to crush or unrepaired transection injuries. This report presents a transgenic mouse, which differs from previously reported and commercially available mice, in that enhanced yellow fluorescent protein expression (EYFP) is driven by the human thy1 promoter (hThy1). Motor and sensory peripheral nerves in these mice appear a bright yellow-green under fluorescent microscopy. This study tracks nerve regeneration in live animals using a serial imaging system. It also introduces a novel model for examining the clinically relevant nerve-injury paradigms of tibial nerve transection repaired with primary neurorrhaphy or graft, and end-to-side neurorrhaphy. Live-animal serial nerve imaging is compared with wet-mount fluorescent microscopy and histomorphometry in the same nerve specimens. The use of transgenic mice that strongly express EYFP in their peripheral neurons, coupled with serial nerve imaging, provide an important methodology for studying the heterogeneous nature of axonal elongation following peripheral-nerve injuries.

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PubMed2004

Effect of perineurial window size on nerve regeneration, blood-nerve barrier integrity, and functional recovery.

End-to-side neurorrhaphy is used clinically to reconstruct nerve injuries when the lack of a suitable proximal nerve stump precludes conventional approaches to microsurgical repair. In end-to-side neurorrhaphy, the distal stump of a transected nerve is sutured to the side of an intact nerve that serves as an axon donor. Prior studies suggest that this perineurial window is a prerequisite for effective nerve regeneration into the recipient nerve. However, the optimal size for this perineurial window remains uncertain. This study evaluated the effect of perineurial window size on collateral axonal sprouting, blood-nerve barrier architecture, and functional impairment of the donor nerve. One hundred twenty Lewis rats were randomized to 1 and 5 mm perineurial window groups and examined at serial time points. The 5 mm perineurial window group exhibited significantly greater fiber counts at the repair zone than the 1mm group within 4 weeks (p < 0.005). Marked breakdown of the blood-nerve barrier was present 2 week postoperatively and resolved by 4 weeks regardless of 1 versus 5 mm perineurial window size. Tibial function indices in both groups normalized between 4 and 6 weeks postoperatively. A large (5 mm) perineurial window induced greater collateral sprouting or regenerative response than a small (1 mm) perineurial window without increasing cross sectional nerve injury or delaying functional recovery.

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PubMed2004

Delayed nerve repair is associated with diminished neuroenhancement by FK506.

OBJECTIVES/HYPOTHESIS: The immunosuppressive agent FK506 has been shown in many studies to enhance nerve regeneration and to accelerate functional recovery after immediate nerve repair. However, in clinical practice the diagnosis and treatment of patients with peripheral nerve injuries is often delayed. The study investigated whether FK506 would retain its neuroregenerative properties when nerve repair and initiation of FK506 therapy were delayed for 7 days. STUDY DESIGN: In vivo laboratory study. METHODS: Thirty-two Lewis rats underwent tibial nerve transection and were randomly assigned to four experimental groups: immediate repair with FK506 treatment, immediate repair without FK506 treatment, 7-day delayed repair with FK506 treatment, and 7-day delayed repair without FK506 treatment. Treated animals received daily subcutaneous injections of 2 mg/kg FK506. Serial walking track measurements were performed at 14, 16, and 18 days after nerve repair. On day 18 after repair, peripheral nerves were injected with a fluorescent tracer for retrograde labeling. On day 21, peripheral nerves and spinal cords were harvested for histomorphometric analysis and motor neuron cell body counts, respectively. RESULTS: Animals that underwent immediate repair with FK506 had significantly higher fiber counts and percentages of nerve than the other three groups (P <.05) but did not show statistically significant earlier functional recovery. The remaining three groups had intermediate levels of nerve regeneration that were not significantly different. Retrograde abled motor neurons counts were decreased in animals with delayed nerve repair that received no FK506 (P <.05). CONCLUSION: In a rat tibial nerve transection model, the neuroregenerative effects of FK506 diminished markedly when repair and initiation of FK506 therapy were delayed by 7 days.

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PubMed2004

Effects of external beam radiation in the rat tibial nerve after crush, transection and repair, or nerve isograft paradigms.

INTRODUCTION: In head and neck surgery, radiation therapy is often administered to an injured nerve. Previous studies have examined the effects of either preoperative or postoperative radiation on nerve regeneration in rodents. In these studies, histomorphometric analysis was performed up to 8 month postoperatively. Given the exceptional neuroregenerative capacity of rodents, significant differences in nerve regeneration may go undetected if nerves are evaluated at such distant postoperative time points. This study is designed with a more appropriate model and investigates the effects of radiation after three common nerve injury paradigms. METHODS: Sixty-four Lewis rates were randomized to 8 groups corresponding to uninjured, tibial nerve crush, transection and repair, or reconstruction with isografts. Half of the animals in each of these paradigms (n = 8 per group) were treated with 10 Gy of external beam radiation to the site of nerve injury at 7 days postoperatively. On postoperative day 28, functional recovery and histomorphometric assessment was performed. RESULTS: For a given paradigm of nerve injury, no significant differences in nerve fiber number, neural density, neural debris, or fiber width were noted between the control and radiated groups, and radiation did not affect functional recovery. CONCLUSION: Radiation had no discernible effect on nerve regeneration or functional recovery in the rodent nerve injury models studied. All assessments were made at time points suitable for detecting differences in nerve regeneration between groups. These findings suggest that administration of radiation to fields containing injured peripheral nerve is unlikely to adversely affect functional outcomes.

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PubMed2004

Anti-CD40 ligand antibody permits regeneration through peripheral nerve allografts in a nonhuman primate model.

Systemic immunosuppression is typically required to prevent allograft rejection. Antibody-based therapies that induce immune unresponsiveness represent an appealing alternative to nonspecific immunosuppression, which is often associated with significant morbidity. In mice, successful prevention of nerve allograft rejection has been demonstrated through interference with the CD40/CD40 ligand interaction. This study investigated the effectiveness of anti-CD40 ligand monoclonal antibody as single-agent therapy in preventing rejection and supporting nerve regeneration across long nerve allografts in nonhuman primates. Twelve outbred cynomolgus macaques were arranged into six genetically mismatched pairs, with each animal receiving a 5-cm ulnar nerve allograft in the right arm and a 5-cm autograft in the left arm. Mixed lymphocyte reaction assays were used to assess resulting immune unresponsiveness. Treated animals (n = 10) received anti-CD40 ligand monoclonal antibody 10 mg/kg one time, locally applied, and 20 mg/kg systemically on postoperative days 0, 1, 3, 10, 18, and 28, and then monthly. Untreated animals (n = 2) served as the untreated controls. At 4 or 6 months after transplantation, nerves were harvested for histological analysis. Four treated animals underwent an additional challenge after cessation of anti-CD40 ligand monoclonal antibody therapy and nerve graft harvests. Autogenous and allogeneic skin and nerve inlay grafting was performed to assess the permanence of immune unresponsiveness induced by anti-CD40 ligand monoclonal antibody. Animals that received anti-CD40 ligand monoclonal antibody demonstrated robust regeneration across nerve allografts, similar to that seen in the autograft control in the contralateral arm. The histomorphometric analysis of allografts in the untreated animals demonstrated significantly worse measurements compared with their matched autograft controls. Animals that received anti-CD40 ligand monoclonal antibody with concomitant skin allografts had virtually no evidence of nerve regeneration through allografts. Allogeneic skin and nerve allografts applied 2 to 12 months after withdrawal of anti-CD40 ligand monoclonal antibody therapy were consistently rejected. This study demonstrates that anti-CD40 ligand monoclonal antibody prevents rejection and allows regeneration of peripheral nerve allografts in nonhuman primates. The effect of anti-CD40 ligand monoclonal antibody appears to be transient, however, with restoration of immunocompetence shortly after withdrawal of therapy.

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PubMed2004

Effect of tension on nerve regeneration in rat sciatic nerve transection model.

Excessive tension across a nerve repair is known to impair nerve regeneration. However, it is uncertain whether nerve grafting is necessary when end-to-end repair would result in only mild to moderate tension. This study investigated the effect of tension on nerve regeneration. Sciatic nerves of 48 Lewis rats were transected and then repaired primarily after resection of 0-, 3-, 6-, or 9-mm lengths of nerve. Postoperative tension levels were quantified using a tensometer. Robust nerve regeneration was observed at 4 weeks in all except the 9-mm repair group, which showed lower nerve fiber counts, percent neural tissue, and nerve density (P < 0.05) and decreased functional recovery. These data indicate that modest levels of tension are well tolerated, but nerve regeneration drops precipitously once a critical tension threshold is exceeded. This threshold was between 0.39 and 0.56 N in the model studied, corresponding to a nerve defect between 6 mm and 9 mm.

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PubMed2003

Use of mixed lymphocyte reaction to identify subimmunosuppressive FK-506 levels in mice.

The immunosuppressive agent FK-506 has a well-described neuroregenerative effect that is mediated by a mechanism independent of calcineurin inhibition. FK-506 levels that fall below the threshold for immunosuppression could therefore potentially enhance nerve regeneration while minimizing toxicity. The purpose of this study was to characterize the dose-dependent effects of FK506 on T-cell proliferation, and establish a subimmunosuppressive dosing regimen for FK-506 in mice. Forty BALB/cJ mice were randomized to four groups corresponding to 0, 0.25, 0.5, or 1.0 mg/kg/day doses of FK-506. Ten days postoperatively, animals were sacrificed, and mixed lymphocyte reaction assays were performed to quantify the immune response to nerve allografts. Mice receiving 0.25 and 0.5 mg/kg/day of FK-506 exhibited a robust T-cell proliferation response, with stimulation indices approaching those of untreated animals. Mice treated with 1.0 mg/kg/day of FK-506 demonstrated significantly decreased T-cell proliferation. These results establish 0.5 mg/kg/day as an upper limit for subimmunosuppressive FK-506 administration.

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PubMed2003

Controlled release of nerve growth factor enhances sciatic nerve regeneration.

Based on previous studies demonstrating the potential of growth factors to enhance peripheral nerve regeneration, we developed a novel growth factor delivery system to provide sustained delivery of nerve growth factor (NGF). This delivery system uses heparin to immobilize NGF and slow its diffusion from a fibrin matrix. This system has been previously shown to enhance neurite outgrowth in vitro, and in this study, we evaluated the ability of this delivery system to enhance nerve regeneration through conduits. We tested the effect of controlled NGF delivery on peripheral nerve regeneration in a 13-mm rat sciatic nerve defect. The heparin-containing delivery system was studied in combination with three doses of NGF (5, 20, or 50 ng/mL) and the results were compared with positive controls (isografts) and negative controls (fibrin alone, NGF alone, and empty conduits). Nerves were harvested at 6 weeks postoperatively for histomorphometric analysis. Axonal regeneration in the delivery system groups revealed a marked dose-dependent effect. The total number of nerve fibers at both the mid-conduit level and in the distal nerve showed no statistical difference for NGF doses at 20 and 50 ng/mL from the isograft (positive control). The results of this study demonstrate that the incorporation of a novel delivery system providing controlled release of growth factors enhances peripheral nerve regeneration and represents a significant contribution toward enhancing nerve regeneration across short nerve gaps.

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PubMed2003

Functional recovery and histomorphometric assessment following tibial nerve injury in the mouse.

Longitudinal studies have established that functional recovery following sciatic nerve injury can be evaluated in the mouse. Injury to the tibial nerve offers several advantages to sciatic nerve injury, including improved lower extremity sensation and end-organ reinnervation. Functional recovery following tibial nerve crush injury was studied in 55 C3H mice randomized into five groups harvested for histomorphometric evaluation from either normal nerves or 2, 3, 4, or 6 weeks postoperatively. Walking tracks were obtained preoperatively, and at regular intervals postoperatively, and foot print lengths measured. Significant normalization of print length occurred 14 days postoperatively, and complete recovery was noted 28 days postoperatively. Significant histomorphologic evidence of neuroregeneration was detected between 2 and 4 weeks postoperatively. Injury to the tibial nerve is a viable alternative to the sciatic nerve for studying neural regeneration in mice, and the print length factor can be used to monitor functional recovery in this model.

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PubMed2003

Effects of delaying FK506 administration on neuroregeneration in a rodent model.

FK506 is an immunosuppressant drug that has been shown experimentally to stimulate nerve growth and speed functional recovery, when administered immediately after peripheral nerve injury. However, the clinical scenario of a peripheral nerve injury is often associated with either a delayed diagnosis or reconstruction. The purpose of this study was to determine the efficacy of FK506 on neuroregeneration with delayed administration. Thirty-two Lewis rats underwent tibial nerve transection with immediate repair. Animals were left untreated, or were treated with daily injections of FK506 (2 mg/kg), started on the day of surgery, postoperative day 3, or postoperative day 5. Animals underwent walking track analysis to assess functional nerve recovery. Nerves were harvested for histomorphometric analysis on postoperative days 21, 28, and 42. Histomorphometry demonstrated that all treatment groups, regardless of the time of drug initiation, demonstrated evidence of enhanced neuroregeneration, compared to the untreated group. Histomorphometric data from groups harvested on day 21 demonstrated a statistically significant improvement in neuroregeneration in the immediate and 3-day delay groups. Therefore, the beneficial effects of FK506 on neuroregeneration are not restricted to immediate administration, but these effects significantly diminish when FK506 is administered 3 days after nerve injury.

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PubMed2003

Dose-dependent effects of FK506 on neuroregeneration in a rat model.

This study explored the effects of different doses of FK506 on peripheral nerve regeneration, to determine whether neuroregeneration could be enhanced without the toxicity of systemic immunosuppression. In the first part of the study, subimmunosuppressive doses of FK506 were determined by examining skin allograft survival in a rat model. Full-thickness skin grafts (2 cm2) from Wistar rats were grafted to recipient Lewis rats. The procedure was performed for six groups (n = 6). The control group received no FK506, and the other five groups received daily doses of FK506 of 0.125, 0.25, 0.5, 1.0, or 2.0 mg/kg. Animals that received 2.0 mg/kg FK506 per day exhibited complete skin graft take, whereas all other groups demonstrated complete rejection. After determination of the immunosuppressive dose of FK506, the neuroregenerative effects of different doses of FK506 were explored by assessing nerve regeneration in 80 rats after tibial nerve transection and repair. The control group received no FK506, whereas the other four groups were given daily doses of FK506 of 0.25, 0.5, 1.0, or 2.0 mg/kg. Rats were euthanized at three time points (25, 30, and 35 days), to fully investigate the effects of different FK506 dosing regimens on neuroregeneration. Histomorphometric analyses performed on postoperative days 30 and 35 demonstrated statistically significant improvements in neuroregeneration with subimmunosuppressive FK506 doses of 0.5 and 1.0 mg/kg per day. Therefore, the study demonstrated that neuroregeneration was enhanced at low doses of FK506 that were not sufficient to prevent skin allograft rejection.

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PubMed2002

Peripheral nerve regeneration in the apolipoprotein-E-deficient mouse.

Apolipoprotein E (apo E) is thought to mediate the reutilization of myelin cholesterol for nerve regeneration. Prior research suggests that apo E is not essential for nerve regeneration following a nerve crush injury. This study was conducted to determine if apo E is essential for nerve regeneration after nerve transection and interposition nerve autograft. Nerve regeneration of transgenic apo E-deficient mice was compared with control mice after a sciatic nerve neurolysis and repair and interposition autograft. Histomorphometric assessment and histology were performed on distal nerve segments to evaluate nerve regeneration. Apo E-deficient mice demonstrated no difference in total fiber number or nerve fiber width when compared with controls; however, the nerve fiber density and percent neural tissue of apo E-deficient mice were significantly less than controls following nerve repair. Apo E deficiency does not affect nerve regeneration. It is likely that the low nerve fiber density and the low percent neural tissue associated with apo E-deficiency result from impairment in the disposal of myelin debris.

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PubMed2002

Effects of tobacco smoke on recovery after nerve crush injury in rats.

The effects of tobacco smoke on nerve healing after crush injury was studied in a rat model. Thirty-six animals were randomized equally into smoke-exposed or nonsmoke-exposed groups. Two nerve crush injuries were performed on the right posterior tibial nerve of each animal at 4-week intervals to mimic a sustained or chronic nerve injury. Recovery of the two groups was assessed with walking track analysis and nerve histomorphometry. There was no difference in the rate of nerve recovery based on walking track analysis in the smoke-exposed animals compared with nonexposed animals. The study also failed to demonstrate any significant difference between the two groups as assessed by histomorphometric criteria.

Analysis of Variance↗

PubMed2002

Failure of cyclosporin a to rescue peripheral nerve allografts in acute rejection.

Prevention and control of graft rejection remain essential in the investigation of peripheral nerve allotransplantation. Although use of cyclosporin A (CsA) has been shown to suppress successfully the rejection of nerve allografts, limited information exists concerning use of this drug to arrest rejection in progress, and thereby effect salvage of these grafts. The aim of this study was to determine the efficacy of CsA in the treatment of ongoing acute rejection of peripheral nerve allografts. Buffalo rats received posterior tibial nerve grafts from either Lewis or Buffalo donor animals and were divided into five groups: group 1 received isografts and no CsA treatment (n = 8), group 2 received allografts with continuous CsA therapy (n = 10), group 3 received allografts with no treatment (n = 7), group 4 received allografts with initiation of CsA therapy delayed until 3 weeks after the procedure (n = 11), and group 5 received allografts with an interrupted course of CsA (n = 15). All grafts were harvested at 10 weeks. Histomorphometric analysis demonstrated comparable nerve regeneration in groups 1 and 2 and good regeneration in group 3 animals, despite cellular infiltrate suggestive of rejection. At 3 weeks after surgery, group 4 animals showed early rejection and significantly less neuroregeneration than positive controls at 10 weeks after delayed initiation of CsA therapy. Finally, group 5 animals showed early regeneration at 3 weeks but significantly lesser regeneration by 10 weeks after interruption of therapy. In this experimental protocol, CsA was ineffective in rescuing histologically proven rejection in progress.

Analysis of Variance↗

PubMed2002

The effects of rapamycin in murine peripheral nerve isografts and allografts.

The FKBP-12-binding ligand FK506 has been successfully used to stimulate nerve regeneration and prevent the rejection of peripheral nerve allografts. The immunosuppressant rapamycin, another FKBP-12-binding ligand, stimulates axonal regeneration in vitro, but its influence on nerve regeneration in peripheral nerve isografts or allografts has not been studied. Sixty female inbred BALB/cJ mice were randomized into six tibial nerve transplant groups, including three isograft and three allograft (C57BL/6J) groups. Grafts were left untreated (groups I and II), treated with FK506 (groups III and IV), or treated with rapamycin (groups V and VI). Nerve regeneration was quantified in terms of histomorphometry and functional recovery, and immunosuppression was confirmed with mixed lymphocyte reactivity assays. Animals treated with FK506 and rapamycin were immunosuppressed and demonstrated significantly less immune cell proliferation relative to untreated recipient animals. Although every animal demonstrated some functional recovery during the study, animals receiving an untreated peripheral nerve allograft were slowest to recover. Isografts treated with FK506 but not rapamycin demonstrated significantly increased nerve regeneration. Nerve allografts in animals treated with FK506, and to a lesser extent rapamycin, however, both demonstrated significantly more nerve regeneration and increased nerve fiber widths relative to untreated controls. The authors suggest that rapamycin can facilitate regeneration through peripheral nerve allografts, but it is not a neuroregenerative agent in this in vivo model. Nerve regeneration in FK506-treated peripheral nerve isografts and allografts was superior to that found in rapamycin-treated animals. Rapamycin may have a role in the treatment of peripheral nerve allografts when used in combination with other medications, or in the setting of renal failure that often precludes the use of calcineurin inhibitors such as FK506.

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PubMed2002

Axonal regeneration after cold preservation of nerve allografts and immunosuppression with tacrolimus in mice.

OBJECT: The purpose of this study was to combine the immunosuppressive and neuroregenerative effects of tacrolimus (FK506) with cold preservation of peripheral nerve allografts to maximize axonal regeneration across short peripheral nerve gaps. METHODS: Ninety-six male C3H mice were randomized to six groups, which were composed of animals with isografts (Group 1, positive control), allografts (Group 2, negative control), allografts treated with subtherapeutic doses of FK506 without and with cold preservation (Groups 3 and 4), and allografts treated with therapeutic doses of FK506 without and with cold preservation (Groups 5 and 6). Results were determined using walking-track data and histomorphometric measurements. Three weeks postoperatively, animals treated with therapeutic doses of FK506 after receiving cold-preserved allografts demonstrated accelerated functional recovery relative to all other groups. In addition, histomorphometric parameters in these animals (1,257 +/- 847 total axons, 6.7 +/- 3.3% nerve tissue, 11.8 +/- 6.5% neural debris, 8,844 +/- 4,325 fibers/mm2 nerve density, and 2.53 +/- 0.25 microm fiber width) were the same as or better than in all other groups. The parameters of percent nerve tissue (p < 0.016), nerve density (p < 0.038), and percent neural debris (p < 0.01) were statistically significantly better than those in all other groups, including Group 1 (isograft, positive control). CONCLUSIONS: The combination of FK506 treatment with cold preservation of nerve allografts resulted in functional and histomorphometric recovery superior to that with either modality alone.

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