Use of ebselen as a neuroprotective agent in rat spinal cord subjected to traumatic injury.

Spinal cord injury (SCI) causes disturbances of motor skills. Free radicals have been shown to be essential for the development of spinal cord trauma. Despite some progress, until now no effective pharmacological therapies against SCI have been verified. The purpose of our experiment was to investigate the neuroprotective effects of ebselen on experimental SCI. Twenty-two rats subjected to SCI were randomly subjected to SCI with no further treatment (n = 10) or intragastric administration of ebselen (10 mg/kg) immediately and 24 hours after SCI. Behavioral changes were assessed using the Basso, Beattie, and Bresnahan locomotor scale and footprint test during 12 weeks after SCI. Histopathological and immunohistochemical analyses of spinal cords and brains were performed at 12 weeks after SCI. Magnetic resonance imaging analysis of spinal cords was also performed at 12 weeks after SCI. Rats treated with ebselen presented only limited neurobehavioral progress as well as reduced spinal cord injuries compared with the control group, namely length of lesions (cysts/scars) visualized histopathologically in the spinal cord sections was less but cavity area was very similar. The same pattern was found in T2-weighted magnetic resonance images (cavities) and diffusion-weighted images (scars). The number of FluoroGold retrogradely labeled neurons in brain stem and motor cortex was several-fold higher in ebselen-treated rats than in the control group. The findings suggest that ebselen has only limited neuroprotective effects on injured spinal cord. All exprimental procedures were approved by the Local Animal Ethics Committee for Experiments on Animals in Katowice (Katowice, Poland) (approval No. 19/2009).

Extended magnetic resonance imaging studies on the effect of classically activated microglia transplantation on white matter regeneration following spinal cord focal injury in adult rats.

Spinal cord injuries are still a serious problem for regenerative medicine. Previous research has demonstrated that activated microglia accumulate in spinal lesions, influencing the injured tissues in various ways. Therefore, transplantation of activated microglia may have a beneficial role in the regeneration of the nervous system. The present study examined the influence of transplanted activated microglial cells in adult rats with injured spinal cords. Rats were randomly divided into an experimental (M) and control (C) group, and were subjected to non-laminectomy focal injury of spinal cord white matter by means of a high-pressured air stream. In group M, activated cultured microglial cells were injected twice into the site of injury. Functional outcome and morphological features of regeneration were analyzed during a 12-week follow-up. The lesions were characterized by means of magnetic resonance imaging (MRI). Neurons in the brain stem and motor cortex were labeled with FluoroGold (FG). A total of 12 weeks after surgery, spinal cords and brains were collected and subjected to histopathological and immunohistochemical examinations. Lesion sizes in the spinal cord were measured and the number of FG-positive neurons was counted. Rats in group M demonstrated significant improvement of locomotor performance when compared with group C (P<0.05). MRI analysis demonstrated moderate improvement in water diffusion along the spinal cord in the group M following microglia treatment, as compared with group C. The water diffusion perpendicular to the spinal cord in group M was closer to the reference values for a healthy spinal cord than it was in group C. The sizes of lesions were also significantly smaller in group M than in the group C (P<0.05). The number of brain stem and motor cortex FG-positive neurons in group M was significantly higher than in group C. The present study demonstrated that delivery of activated microglia directly into the injured spinal cord gives some positive effects for the regeneration of the white matter.

Sciatic nerve regeneration in rats subjected to ketogenic diet.

OBJECTIVES: Ketogenic diet (KD) is a high-fat-content diet with insufficiency of carbohydrates that induces ketogenesis. Besides its anticonvulsant properties, many studies have shown its neuroprotective effect in central nervous system, but its influence on peripheral nervous system has not been studied yet. We examined the influence of KD on regeneration of peripheral nerves in adult rats. METHODS: Fifty one rats were divided into three experimental (n = 15) and one control (n = 6) groups. Right sciatic nerve was crushed and animals were kept on standard (ST group) or ketogenic diet, the latter was introduced 3 weeks before (KDB group) or on the day of surgery (KDA group). Functional (CatWalk) tests were performed once a week, and morphometric (fiber density, axon diameter, and myelin thickness) analysis of the nerves was made after 6 weeks. Body weight and blood ketone bodies level were estimated at the beginning and the end of experiment. RESULTS: Functional analysis showed no differences between groups. Morphometric evaluation showed most similarities to the healthy (uncrushed) nerves in KDB group. Nerves in ST group differed mostly from all other groups. Ketone bodies were elevated in both KD groups, while post-surgery animals' body weight was lower as compared to ST group. DISCUSSION: Regeneration of sciatic nerves was improved in KD - preconditioned rats. These results suggest a neuroprotective effect of KD on peripheral nerves.

Grafted Activated Schwann Cells Support Survival of Injured Rat Spinal Cord White Matter.

BACKGROUND AND OBJECTIVE: The influence of cultured Schwann cells on injured spinal cord in rats is examined. METHODS: Focal injury of spinal cord white matter at the T10 level was produced using our original non-laminectomy method with a high-pressure air stream. Schwann cells from 7-day predegenerated rat sciatic nerves were cultured, transducted with green fluorescent protein and injected into the cisterna magna (experimental group) 3 times: immediately after spinal cord injury and 3 and 7 days later. Neurons in the brainstem and motor cortex were labeled with FluoroGold (FG) delivered caudally from the injury site a week before the end of the experiment. The functional outcome and morphologic features of neuronal survival were analyzed during a 12-week follow-up. The lesions were visualized and analyzed using magnetic resonance imaging. The maximal distance of expansion of implanted cells in the spinal cord was measured and the number of FG-positive neurons in the brain was counted. RESULTS: Rats treated with Schwann cells presented significant improvement of locomotor performance and spinal cord morphology compared with the control group. The distance covered by Schwann cells was 7 mm from the epicenter of the injury. The number of brainstem and motor cortex FG-positive neurons in the experimental group was significantly higher than in the control group. CONCLUSIONS: The data show that activated Schwann cells are able to induce the repair of injured spinal cord white matter. The route of application of cells via the cisterna magna seemed to be useful for their delivery in spinal cord injury therapy.

Surgical treatment of subependymal giant cell astrocytoma in tuberous sclerosis complex patients.

BACKGROUND: Subependymal giant cell astrocytoma is a brain tumor associated with tuberous sclerosis complex. There are two treatment options for subependymal giant cell astrocytomas: surgery or mammalian target of rapamycin inhibitor. The analysis of outcome of subependymal giant cell astrocytoma surgery may help characterize the patients who may benefit from pharmacotherapy. METHODS: Sixty-four subependymal giant cell astrocytoma surgeries in 57 tuberous sclerosis complex patients with at least a 12-month follow-up were included in the study. The tumor size, age of the patients, mutation in the TSC1 or TSC2 gene, indication for the surgery, and postsurgical complications were analyzed. RESULTS: The mean age of patients at surgery was 9.7 years. Mean follow-up after surgery was 63.7 months. Thirty-seven (57.8%) tumors were symptomatic and 27 (42.2%) were asymptomatic. Patients with TSC2 mutations developed subependymal giant cell astrocytoma at a significantly younger age than individuals with TSC1 mutations. Four patients (6.2% of all surgeries) died after surgery. Surgery-related complications were reported in 0%, 46%, 83%, 81%, and 67% of patients with tumors <2 cm, between 2 and 3 cm, between 3 and 4 cm, >4 cm, and bilateral subependymal giant cell astrocytomas, respectively, and were most common in children younger than 3 years of age. The most common complications included hemiparesis, hydrocephalus, hematoma, and cognitive decline. CONCLUSIONS: Our study indicates that subependymal giant cell astrocytoma surgery is associated with significant risk in individuals with bilateral subependymal giant cell astrocytomas, tumors bigger than 2 cm, and in children younger than 3 years of age. Therefore, tuberous sclerosis complex patients should be thoroughly screened for subependymal giant cell astrocytoma growth, and early treatment should be considered in selected patients.

Air gun impactor--a novel model of graded white matter spinal cord injury in rodents.

Understanding mechanisms of spinal cord injury and repair requires a reliable experimental model. We have developed a new device that produces a partial damage of spinal cord white matter by means of a precisely adjusted stream of air applied under high pressure. This procedure is less invasive than standard contusion or compression models and does not require surgical removal of vertebral bones. We investigated the effects of spinal cord injury made with our device in 29 adult rats, applying different experimental parameters. The rats were divided into three groups in respect to the applied force of the blast wave. Functional outcome and histopathological effects of the injury were analyzed during 12-week follow-up. The lesions were also examined by means of magnetic resonance imaging (MRI) scans. The weakest stimulus produced transient hindlimb paresis with no cyst visible in spinal cord MRI scans, whereas the strongest was associated with permanent neurological deficit accompanied by pathological changes resembling posttraumatic syringomyelia. Obtained data revealed that our apparatus provided a spinal cord injury animal model with structural changes very similar to that present in patients after moderate spinal cord trauma.

Interleukin-1ß increases release of endothelin-1 and tumor necrosis factor as well as reactive oxygen species by peripheral leukocytes during experimental subarachnoid hemorrhage.

In the subarachnoid hemorrhage (SAH) blood mixes with cerebrospinal fluid, what starts immunoinflammatory processes - increased synthesis of proinflammatory cytokines, and formation of reactive oxygen species (ROS), resulting in pre-activation of different populations of peripheral leukocytes. Migration of leukocytes to the brain parenchyma through broken blood brain barrier may produce extra brain tissue injury besides of that resulting from SAH. We examined in adult rats the effect of interleukin-1ß (IL-1ß) neutralization on secretion of cytokines as well as production of ROS in the course of SAH. SAH was produced by injection of 150 µL of autologous arterial blood into cisterna magna. In 50% of animals, IL-1beta activity was inhibited by intracerebroventricular administration of anti-rat IL-1ß antibodies. Ninety minutes or 24 hrs following surgery, blood samples were drawn from the extraorbital plexus and centrifuged to obtain two leukocyte subpopulations - polymorphonuclear (PMN) and mononuclear (MN). The chemiluminescence, a hallmark of ROS synthesis, was measured in PMNs. In supernatants from MNs cultures, concentrations endothelin-1 (ET-1), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were assessed. SAH caused the increase ofn PMNs chemiluminescence as well as the increase of production of ET-1 and TNF-α by MNs but had no influence on IL-6 concentration. Neutralization of IL-1ß resulted in significant decrease of chemiluminescence as well as concentration of both ET-1 and TNF-α, while IL-6 concentration was increased. These revealed an important role of IL-1ß in the activation of peripheral leukocytes in the course of subarachnoid hemorrhage.

Reduction of post-traumatic neuroma and epineural scar formation in rat sciatic nerve by application of microcrystallic chitosan.

Injury of peripheral nerve is associated with the development of post-traumatic neuroma at the end of the proximal stump, often being the origin of neuropathic pain. This type of pain is therapy-resistant and therefore extremely nagging for patients. We examined the influence of the microcrystallic chitosan gel applied to the proximal stump of totally transected sciatic nerve on the neuroma formation and neuropathic pain development in rats. In 14 rats, right sciatic nerve was transected and the distal stump was removed to avoid spontaneous rejoining. In the chitosan (experimental) group (n = 7), the proximal stump was covered with a thin layer of the microcrystallic chitosan gel. In control animals (n = 7), the cut nerve was left unsecured. Autotomy, an animal model of neuropathic pain, was monitored daily for 20 weeks following surgery. Then, the animals were perfused transcardially and the proximal stumps of sciatic nerves were dissected and subjected to histologic evaluation. The presence, size, and characteristics of neuromas as well as extraneural fibrosis were examined. In chitosan group, the incidence and the size of the neuroma were markedly reduced, as compared with the control group; however, there was no difference in autotomy behavior between groups. In addition, extraneural fibrosis was significantly reduced in chitosan group when compared to the control group. The results demonstrate beneficial influence of microcrystallic chitosan applied to the site of nerve transection on the development of post-traumatic neuroma and reduction of extraneural fibrosis, however without reduction of neuropathic pain.

APP/SOD1 overexpressing mice present reduced neuropathic pain sensitivity.

There are controversies regarding pain expression in mentally disabled people, including Down syndrome patients. The aim of this study was to examine neuropathic pain-related behavior and peripheral nerve regeneration in mouse model of Down syndrome. Sciatic nerves of double transgenic mice, overexpressing both amyloid precursor protein (APP) and Cu/Zn superoxide dismutase (SOD1) genes, and FVB/N wild type mice were transected and immediately resutured. Evaluation of autotomy and functional recovery was carried out during 4-week follow-up. We found markedly less severe autotomy in transgenic animals, although the onset of autotomy was significantly delayed in control mice. Interestingly, neuroma formation at the injury site was significantly more prominent in transgenic animals. Sciatic function index outcome was better in transgenic mice than in wild-type group. Histological evaluation revealed no statistically significant differences in the number of GAP-43-positive growth cones and macrophages in the distal stump of the transected nerve between groups. However, in transgenic animals, the regenerating axons were arranged more chaotically. The number of Schwann cells in the distal stump of the transected nerves was significantly lower in transgenic mice. The number of surviving motoneurons was markedly decreased in transgenic group. We measured also the atrophy of denervated muscles and found it decreased in APP/SOD1 overexpressing mice. Taken together, in this model of Down syndrome, we observed increased neuroma formation and decreased autotomy after peripheral nerve injury. Our findings suggest that APP/SOD1 overexpressing mice are less sensitive for neuropathic pain associated with neuroma.

Interleukin-1beta plays a role in the activation of peripheral leukocytes after blood-brain barrier rupture in the course of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) develops when extravasated arterial blood enters subarachnoid space and mixes with cerebrospinal fluid. As a result, many pathologies develop, including increase in brain-blood barrier (BBB) permeability and activation of peripheral leukocytes, that in turn augments immuno-inflammatory response, considered as the cause of numerous complications following SAH. In the study, we examined the role of one of major cytokines, interleukin 1-beta (IL-1beta), in the BBB rupture and subsequent migration of leukocytes into central nervous system (CNS) after experimental SAH in adult rats. SAH was produced by injection of 150 uL of autologous arterial blood into cisterna magna. In 50% of animals, IL-1beta activity was inhibited by intracerebroventricular administration of anti-rat IL-1beta antibodies (SAH' groups). Control group consisted of sham-operated rats. Ninety minutes or 24 hrs following surgery, blood samples were taken, then animals were perfused transcardially and whole brains were collected. Three major populations of leukocytes present at brain stem and frontal part of the brain - granulocytes, monocytes/macrophages and lymphocytes - were labeled with appropriate antibodies. S-100B protein concentration in the serum, a marker of BBB permeability, was also measured. Neutralization of IL-1beta activity reduced elevated S-100B level and number of leukocytes found within the CNS, however these changes were not uniformly significant among different subgroups. The results demonstrate an important role of IL-1beta in the BBB damage and leukocyte migration into the CNS subarachnoid hemorrhage.

APP overexpression prevents neuropathic pain and motoneuron death after peripheral nerve injury in mice.

Despite general capacity of peripheral nervous system to regenerate, peripheral nerve injury is often followed by incomplete recovery of function and sometimes burdened by neuropathic pain. Amyloid precursor protein (APP) was suggested to play a role in neuronal growth, however, its role in peripheral nerve repair was not studied. The aim of this study was to examine the role of APP overexpression in peripheral nerve regeneration and neuropathic pain-related behavior in mice. Sciatic nerves of APP overexpressing and FVB/N wild-type mice were transected and immediately resutured. Evaluation of motor and sensory function and autotomy was carried out during 4-week follow up. We found no autotomy behavior as well as less significant atrophy of denervated muscles in APP overexpressing animals when compared to wild-type ones. Sciatic nerve function index outcome did not differ between groups. Histological evaluation revealed that the intensity of regeneration features, including GAP-43-positive growth cones and Schwann cells number in the distal stump of the transected nerve, was also similar in both groups. However, the regenerating fibers were organized more chaotically in wild-type mice and neuromas were much more often seen in this group. The number of macrophages infiltrating the injury site was significantly higher in control group. The number of surviving motoneurons was higher in transgenic mice than in control animals. Taken together, our findings suggest that APP overexpression is beneficial for nerve regeneration processes due to better organization of regenerating fibers, increased survival of motoneurons after autotomy and prevention of neuropathic pain.

17beta-estradiol and predegenerated nerve graft effect on hippocampal neurogenesis in adult female rats.

OBJECTIVES: The aim of present work was to examine estrogen influence on neurogenesis in the model of predegenerated peripheral nerve grafts implantation into the rat hippocampal dentate gyrus. METHODS: Experiment was carried out on female rats divided into three experimental groups: NO - non-ovariectomized, OV - ovariectomized and E - heterogeneous group with various 17-beta-estradiol substitution after ovariectomy. Proliferating cells were labeled with BrdU. Brains were subjected to immunohistochemical procedures to visualize nestin, GFAP and estrogen receptors (ERalpha and ERbeta). RESULTS: Proliferation rate was highest in E groups with estrogen levels resembling that in proestrus phase. Ovariectomy resulted in higher than in NO group number of new neurons, while high hyperestrogenemia worsened the results. The proportions of nestin-labeled cells correlated in similar way with different hormonal state. We found also distinct co-localization of nestin and GFAP in E group (proestrus). It may suggest the presence of radial glia, a potential source of new neurons in adult mammals. Nerve graft induced ERalpha expression at the site of injury in all groups. Distribution of ERbeta in hippocampus was estradiol-dose-dependent and correlated with cell proliferation. CONCLUSION: In our model, 17-beta-estradiol and predegenerated nerve graft implantation had synergistic effect on hippocampal neurogenesis.

Neutralization of interleukin-1beta reduces vasospasm and alters cerebral blood vessel density following experimental subarachnoid hemorrhage in rats.

Subarachnoid hemorrhage (SAH) develops when extravasated arterial blood enters subarachnoid space and mixes with cerebrospinal fluid. As a result, many pathologies develop, including arterial vasospasm that leads to the ischemia and hypoxia. Immuno-inflammatory response is considered as the cause of numerous complications following SAH. In the study, we examined the role of one of major cytokines, interleukin 1-beta (IL-1beta), on the vascular pathologies after experimental SAH in adult rats. SAH was produced by injection of 150 uL of autologous arterial blood into cisterna magna. In 50% of animals, IL-1beta activity was inhibited by intracerebroventricular administration of anti-rat IL-1beta antibodies (SAH' groups). Control group consisted of sham-operated rats. Ninety minutes or 24 hrs following surgery, animals were perfused transcardially and whole brains were collected. Spasm index (ratio of vessel diameter to the mean wall thickness) of basilar artery as well as blood vessel density (number of vessels per square millimeter) at brain stem and frontal part of the brain were measured. SAH led to the vasospasm of basilar artery and increased the density of blood vessel. Neutralization of IL-1beta activity significantly reduced both the vasospasm and blood vessel density only 24 hrs after SAH. The results demonstrate an important role of IL-1beta in the delayed development of vascular pathologies after subarachnoid hemorrhage.

Cardiac rhabdomyomas in tuberous sclerosis complex show apoptosis regulation and mTOR pathway abnormalities.

Cardiac rhabdomyoma (CR) is the most common heart tumor in children and is usually associated with tuberous sclerosis complex (TSC). Tuberous sclerosis complex is a genetic disorder caused by a mutation in either of 2 genes (TSC1 or TSC2) and characterized by the formation of hamartomas in multiple organs. The 2 TSC proteins, hamartin and tuberin, antagonize the mammalian target of rapamycin (mTOR) signaling pathway, thus regulating cell growth and proliferation. Recently, some trials treating TSC with the mTOR inhibitor rapamycin have been published; however, the impact of such treatment on heart tumors is not known. The aim of the present paper was to study the molecular pathobiology of CRs. Six CR samples were studied. The expression of S6K1, pErk, Erk, Akt, pAkt, 4E-BP1, hamartin, tuberin, mTOR, bcl-2, Bax, and Ki-67 was examined using immunohistochemistry and Western blot methods. Increased expression of Bax, mTOR, pS6K, pErk, and 4E-BP1 was found in all CR samples. Hamartin and tuberin expression was decreased in tumors versus normal heart tissues. This is the first study showing mTOR pathway dysregulation and an increased expression of proapoptotic Bax protein in CRs associated with TSC.

Expression of tuberin and hamartin in tuberous sclerosis complex-associated and sporadic cortical dysplasia of Taylor's balloon cell type.

Focal cortical dysplasia (FCD) type IIB is a malformation of cortical development characterized by presence of balloon cells. These cells share phenotypic features of giant cells found in tuberous sclerosis complex (TSC), but the relationship between FCD type IIB and TSC is not well established. TSC is an autosomal dominant disorder caused by mutation in either of two genes: TSC1, encoding hamartin, and TSC2, encoding tuberin. Both proteins form a complex inhibiting mTOR signalling pathway and thus regulate cell size and proliferation. In this study, tuberin and hamartin expression was evaluated under a confocal microscope in six cases of Taylor's balloon cell type FCD. Three patients met the clinical criteria for TSC. In three other patients, TSC was excluded based on a panel of clinical and radiological examinations. Additionally, two cases of FCD type I and 3 samples of normal brain tissue were used as a reference group. We found loss of tuberin and hamartin expression in FCD type IIB lesions from patients with TSC. In sporadic FCD type IIB cases, only a few tuberin and hamartin positive cells were detected in the white-grey matter junction and in deeper parts of the white matter. Cortical balloon cells showed loss of both tuberin and hamartin. In contrast, the expression of tuberin and hamartin in FCD type I samples was strong, similarly to normal brain tissue. In conclusion, loss of TSC1 and TSC2 products expression in balloon cells of both cortical dysplasia type IIB in TSC-related and sporadic patients suggests that FCD type IIB may represent the focal form of TSC.

The influence of trkB deficiency on long-term outcome of peripheral nerve injury in mice.

The long-term outcome of peripheral nerve injury is often unsatisfactory, especially if the injury resulted in a gap between transected nerve stumps. Brain-derived neurotrophic factor and its receptor, trkB, are strongly implicated in the early phase of axonal regeneration after injury. We examined the role of trkB in long-term functional and morphological outcome of peripheral nerve injury. The sciatic nerve was transected in wild-type and heterozygous trkB-deficient mice. The nerve was either left cut or immediately sewn up or the gap injury model was performed. The gap was provided with autologous or cross (obtained from other genetic group) graft. Sciatic nerve function as well as autotomy was assessed during 16-week follow-up. The long-term functional outcome of nerve cut or immediately rejoined did not differ between wild-type and trkB-deficient mice. Gap injury provided with nerve graft resulted in better functional outcome in trkB-deficient mice than wild-type animals. Sixteen weeks after the surgery, the animals were sacrificed and histological evaluations were performed. The number of nerve fibres regenerating into the distal stump of transected and rejoined nerves did not differ between wild-type and trkB-deficient animals. TrkB deficiency markedly increased the number of Schwann cells as well as mast cells at the injury site and in the distal stump of the regenerating nerve. TrkB deficient nerves also showed higher expression of bcl-2 protein but lower of trkA and NGF than wild-type ones. Our results show for the first time the possible deleterious role of trkB receptor in long-term outcome of peripheral nerve injury.

BDNF contributes to animal model neuropathic pain after peripheral nerve transection.

The outcome of peripheral nerve injury is often impaired by neuropathic pain, which is resistant to most analgesics and presents a serious clinical problem. The mechanisms underlying post-traumatic neuropathic pain remain unclear, but they are likely associated with the regeneration processes. Brain-derived neurotrophic factor (BDNF) is known to enhance peripheral nerve regeneration and is also considered to be an endogenous modulator of nociceptive responses following spinal cord lesion. The aim of this work was to examine the local effect of BDNF in a neuropathic pain model. Sciatic nerves of adult male rats were transected and supplied with connective tissue chambers filled with (1) fibrin only, (2) fibrin with BDNF, or (3) fibrin with antibodies against BDNF. In control animals the nerve was transected and no chamber was applied. During follow-up, autotomy behavior was assessed. Seven weeks after the operation, the number of surviving and regenerating neurons in dorsal root ganglia was counted and the neuroma incidence was examined. We found that local inactivation of BDNF decreased the incidence as well as severity of autotomy and neuroma formation, but did not influence neuron regeneration into the chambers. These results indicate that BDNF plays a locally crucial role in neuropathic pain development after peripheral nerve injury.

TrkB deficiency increases survival and regeneration of spinal motoneurons after axotomy in mice.

Persisting motor function deficit after peripheral nerve injury often results from axotomized motoneuron death. Brain-derived neurotrophic factor (BDNF) and its receptor, trkB, are known to promote peripheral nerve regeneration. However, the requirement of BDNF and trkB for adult motoneuron survival after peripheral nerve injury is not established. We studied the number of surviving and regenerating motoneurons after sciatic nerve transection in wild-type and heterozygous trkB-deficient mice. The nerve was either left cut or immediately sewed up or the gap injury model was performed. The gap was provided with an autologous or cross (obtained from other genetic group) graft. Sixteen weeks after surgery, the animals were sacrificed and histological evaluations were performed. In order to study the number of regenerating motoneurons, immunofluorescent tracer was applied to the distal stump of the operated nerve. We found that in wild type mice, the decrease in motoneurons after nerve transection was markedly higher than in trkB-deficient animals, regardless of the operation procedure. Nerve transection resulted in the highest decrease in motoneuron number in wild type mice. This decrease was lower if the nerve was re-joined using a cross-graft obtained from a trkB-deficient animal. Interestingly, in trkB-deficient animals, the decrease in motoneuron count did not depend on type of operation and was similar after nerve transection, re-joining or grafting. The number of regenerating motoneurons after nerve transection and re-joining in wild type animals was lower than in trkB-deficient mice. The number of regenerating motoneurons after nerve grafting did not differ between groups. These results provide further evidence for the role of trkB receptor in spinal motoneuron survival and regeneration.

Effect of oblique nerve grafting on peripheral nerve regeneration in rats.

Current methods of peripheral nerve repair are to rejoin cut nerve stumps directly or to bridge large gaps with autologous nerve grafts. In both cases the surface of nerve stump endings is typically cut perpendicularly to the long axis of the nerve. The outcome of such operations, however, is still not satisfactory. In this study, we examine the effect of oblique nerve cutting and grafting on morphological as well as functional features of regeneration. In adult rats, sciatic nerve was cut and rejoined either directly or using an autologous graft, at 90 degrees or 30 degrees angle. Functional regeneration was assessed by walking track analysis during 12-week follow-up. Afterwards muscle weight was measured and histological studies were performed. The latter included nerve fibers and Schwann cells counting, as well as visualization of scar formation and epineural fibrosis. Nerves cut obliquely and rejoined showed better functional recovery than perpendicularly transected. Similar effect was observed after oblique grafting when compared to perpendicular one. Numbers of nerve fibers growing into the distal stump of the nerve as well as the number of Schwann cells were significantly higher in obliquely than in perpendicularly operated nerves. Moreover, growing axons were arranged more regularly following oblique treatment. These data indicate that joining or grafting the nerve stumps at acute angle is a more profitable method of nerve repair than the standard procedure performed at right angle.

The role of trkB receptor in the formation of post-traumatic neuroma.

The outcome of peripheral nerve injury is often impaired by post-traumatic neuroma developing at the injury site. Neuroma is usually accompanied by neuropathic pain, which is usually resistant to most analgesics and presents a serious clinical problem. The mechanisms underlying post-traumatic neuroma remain unclear, but they are likely associated with regeneration processes. Brain-derived neurotrophic factor (BDNF) and its receptor, trkB, are strongly implicated in axonal regeneration after injury. The aim of this work was to examine the role of trkB in post-traumatic neuroma formation. The sciatic nerve was transected in wild-type and heterozygous trkB-deficient mice. The nerve was either left cut or immediately sewn up or the gap injury model was performed. The gap was provided with an autologous or cross (obtained from another genetic group) graft. Sixteen weeks after surgery, the animals were sacrificed and histologic evaluations were performed. We found very limited or no neuroma formation in wild-type animals, regardless of the surgical procedure. In the majority of trkB-deficient mice, the post-traumatic neuroma was found at the end of the proximal stump of the transected nerve. In the gap injury model, in trkB-deficient animals receiving wild-type graft, there was no neuroma at the join site between the graft and distal stump of the nerve. In contrast, if the graft was autologous, neuroma formed at both joints. We also noticed many more mast cells accumulated at the surgery site in trkB-deficient than in wild-type animals. These results indicate the important role of BDNF receptor in post-traumatic neuroma formation.