Neurofilament Immunohistochemistry Research Articles

Pentoxifylline as Add-On Treatment to Donepezil in Copper Sulphate-Induced Alzheimer’s Disease-Like Neurodegeneration in Rats

Alzheimer’s disease (AD), the most common neurodegenerative disorder, is characterized by behavioral, cognitive, and progressive memory impairments. Extensive neuronal loss, extracellular accumulation of insoluble senile amyloid-β (Aβ) plaques, and intracellular neurofibrillary tangles (NFTs) are the major pathological features. The present study aimed to investigate the therapeutic effect of donepezil (DON) and pentoxifylline (PTX) in combination to combat the neurodegenerative disorders (experimental AD) induced by CuSO4 intake in experimental rats. Thirty adult male Wistar rats (140–160 g) were used in this study. AD was first induced in rats by CuSO4 supplement to drinking water (10 mg/L) for 14 weeks. The AD group received no further treatment. Oral treatment with DON (10 mg/kg/day), PTX (100 mg/kg/day), or DON + PTX for the other three groups was started from the 10th week of CuSO4 intake for 4 weeks. Cortex markers like acetylcholine (ACh), acetylcholinesterase (AChE), total antioxidant capacity (TAC), and malondialdehyde (MDA) and hippocampus markers like β-amyloid precursor protein cleaving enzyme 1 (BACE1), phosphorylated Tau (p-tau), Clusterin (CLU), tumor necrosis factor-α (TNF-α), caspase-9 (CAS-9), Bax, and Bcl-2 were measured. The histopathology studies were done by using hematoxylin and eosin and Congo red stains as well as immunohistochemistry for neurofilament. CuSO4 induced adverse histological and biochemical changes. The histological injury in the hippocampus was inhibited following the administration of the DON and PTX. The brain tissue levels of AChE, MDA, BACE1, p-tau, CLU, CAS-9, Bax, and TNF-α were significantly increased, while brain tissue levels of ACh, TAC, and Bcl-2 were significantly decreased in CuSO4-treated rats as compared with the untreated control group. The effects induced by either DON or PTX on most studied parameters were comparable. Combined treatment of DON and PTX induced remarkable results compared with their individual use. However, more clinical and preclinical studies are still required to further confirm and prove the long-term efficacy of such combination.

Vitamin D alleviates cognitive dysfunction and brain damage induced by copper sulfate intake in experimental rats: focus on its combination with donepezil.

This study aimed to demonstrate the potential benefits of donepezil (DPZ) and vitamin D (Vit D) in combination to counteract the neurodegenerative disorders induced by CuSO4 intake in experimental rats. Neurodegeneration (Alzheimer-like) was induced in twenty-four male Wistar albino rats by CuSO4 supplement to drinking water (10mg/L) for 14weeks. AD rats were divided into four groups: untreated AD group (Cu-AD) and three treated AD groups; orally treated for 4weeks with either DPZ (10mg/kg/day), Vit D (500IU/kg/day), or DPZ + Vit D starting from the 10th week of CuSO4 intake. Another six rats were used as normal control (NC) group. The hippocampal tissue content of β-amyloid precursor protein cleaving enzyme 1 (BACE1), phosphorylated Tau (p-tau), clusterin (CLU), tumor necrosis factor-α (TNF-α), caspase-9 (CAS-9), Bax, and Bcl-2 and the cortical content of acetylcholine (Ach), acetylcholinesterase (AChE), total antioxidant capacity (TAC), and malondialdehyde (MDA) were measured. Cognitive function tests (Y-maze) and histopathology studies (hematoxylin and eosin and Congo red stains) and immunohistochemistry for neurofilament. Vit D supplementation alleviated CuSO4-induced memory deficits including significant reduction hippocampal BACE1, p-tau, CLU, CAS-9, Bax, and TNF-α and cortical AChE and MDA. Vit D remarkably increased cortical Ach, TAC, and hippocampal Bcl-2. It also improved neurobehavioral and histological abnormalities. The effects attained by Vit D treatment were better than those attained by DPZ. Furthermore, Vit D boosted the therapeutic potential of DPZ in almost all AD associated behavioral and pathological changes. Vit D is suggested as a potential therapy to retard neurodegeneration.

Peripheral neuropathy caused by fenugreek (Trigonella foenum-graecum) straw intoxication in cattle and experimental reproduction in sheep and goats.

Trigonella foenum-graecum (fenugreek) is a legume widely used as a food supplement in humans and less frequently in ruminants. Toxicity has been described sporadically in ruminants grazing mature fenugreek plants or stubble; however, the pathological features are unclear. This report describes a natural outbreak of intoxication in cattle fed fenugreek straw and the experimental reproduction using 8 sheep and 8 goats. Affected cattle presented clinical signs approximately 1 month after consuming the straw and 100 of 400 cattle (25%) were affected, of which 60 of 100 (60%) died or were euthanized. Clinical signs were characterized by proprioceptive positioning defects with abnormal postures and weakness of hindlimbs. Forelimbs were also affected in severely affected animals, and cattle became recumbent. Locomotion was characterized by trembling, and some cattle showed high-stepping movements of their forelimbs and knuckled over in their fetlocks. Experimental intoxication induced clinical signs only in sheep and were similar to cattle, although with signs starting in the forelegs. Gross and microscopic lesions were similar in spontaneous and experimental intoxications. Macroscopic changes corresponded with muscular hemorrhages and edema, mainly surrounding the peripheral nerves. Microscopic examination only demonstrated lesions in the distal peripheral nerves, which included edema, hemorrhages, and Wallerian degeneration. Neurofilament immunohistochemistry revealed altered axon labeling and S100 showed a decrease in myelin intensity and loss of its typical compact arrangement around axons. Biochemical and hematological abnormalities included elevated levels of muscle and liver enzymes and thrombocytopenia. These findings indicate that fenugreek straw induces peripheral neuropathy in cattle and sheep, but not in goats.

Magnetic Resonance Imaging and Micro-Computed Tomography reveal brain morphological abnormalities in a mouse model of early moderate prenatal ethanol exposure

BackgroundThis study investigated the effects of early moderate prenatal ethanol exposure (PEE) on the brain in a mouse model that mimics a scenario in humans, whereby moderate daily drinking ceases after a woman becomes aware of her pregnancy. MethodsC57BL/6J pregnant mice were given 10% v/v ethanol from gestational day 0–8 in the drinking water. The male offspring were used for imaging. Anatomical and diffusion Magnetic Resonance Imaging were performed in vivo at postnatal day 28 (P28, adolescence) and P80 (adulthood). Micro-Computed Tomography was performed on fixed whole heads at P80. Tensor-based morphometry (TBM) was applied to detect alterations in brain structure and voxel-based morphometry (VBM) for skull morphology. Diffusion tensor and neurite orientation dispersion and density imaging models were used to detect microstructural changes. Neurofilament (NF) immunohistochemistry was used to validate findings by in vivo diffusion MRI. ResultsTBM showed that PEE mice exhibited a significantly smaller third ventricle at P28 (family-wise error rate (FWE), p < 0.05). All other macro-structural alterations did not survive FWE corrections but when displayed with an uncorrected p < 0.005 showed multiple regional volume reductions and expansions, more prominently in the right hemisphere. PEE-induced gross volume changes included a bigger thalamus, hypothalamus and ventricles at P28, and bigger total brain volumes at both P28 and P80 (2-sample t-tests). Disproportionately smaller olfactory bulbs following PEE were revealed at both time-points. No alterations in diffusion parameters were detected, but PEE animals exhibited reduced NF positive staining in the thalamus and striatum and greater bone density in various skull regions. ConclusionOur results show that early moderate PEE can cause alterations in the brain that are detectable during development and adulthood.

Neurofilament Immunohistochemistry Followed by Luxol Fast Blue, for Staining Axons and Myelin in the Same Paraffin Section of Spinal Cord.

Many disorders of the central nervous system are characterized by both axonal pathology and demyelination. In assessing this concurrent pathology, techniques for staining axons or myelin are frequently used separately. Here we report the development of a combined immunohistochemical and tinctorial staining technique in which we have modified the Luxol fast blue myelin stain to be used in conjunction with a diaminobenzidine-based immunohistochemical stain for high molecular weight neurofilament (SMI-31). This modification of staining will have utility in experimental neuropathology laboratories investigating demyelination and axonal damage in human tissue and animal models.

Mucosectomy disrupting the enteric nervous system causes contraction and shrinkage of gastrointestinal flaps: potential implications for augmentation cystoplasty

Augmenting the bladder with a seromuscular gastrointestinal flap is a promising alternative approach aiming for a mucus-free bladder augmentation; however, the contraction (shrinkage) of the flaps remains a major concern. Enteric nervous system (ENS) abnormalities cause a failure of relaxation of the intestinal muscle layers in motility disorders such as Hirschsprung's disease and intestinal neuronal dysplasia. In mammals, the submucosal enteric nervous plexus contains nitrergic inhibitory motor neurons responsible for muscle relaxation. The authors hypothesize that mucosectomy disconnects the submucosal nervous plexus from the myenteric plexus resulting in flap shrinkage. After ethical approval, mucosectomy was performed on vascularized flaps from the ileum, colon, and stomach in five anesthetized pigs. In Group (I), only the mucosa was scraped off with forceps, creating a sero-musculo-submucosal flap, while in Group (II), the mucosa and submucosa were peeled off as one layer, leaving a seromuscular flap. Isolated and detubularized segments served as control. The width of each flap was measured before and after the mucosectomy. The ENS was assessed by neurofilament immunohistochemistry in conventional sections and by acetylcholinesterase and NADPH-diaphorase enzyme histochemistry in whole-mount preparations. The stomach contracted to a lesser extent of its original width, 92.82±7.86% in Group (I) and 82.24±6.96% in Group (II). The ileum contracted to 81.68±4.25% in Group (I) and to 72.675±5.36% in Group (II). The shrinkage was most noticeable in the colon: 83.89±15.73% in Group (I) and to 57.13±11.51% in Group (II). One-way equal variance test showed significant difference (P<0,05) between Group (I) and (II), comparing stomach with ileum and ileum with colon. The histochemistry revealed that the submucosal nervous plexus containing nitrergic inhibitory neurons was disconnected from the myenteric plexus in Group (II) of all specimens. Mucosectomy resulted in significant immediate shrinkage of the flaps. This was more expressed when also the submucosa was peeled off, thus fully disrupting the ENS. The shrinkage affected the stomach the least and the colon the greatest. This phenomenon should be taken into consideration when planning mucus-free bladder augmentation.

Neurofilament is superior to cytokeratin 20 in supporting cutaneous origin for neuroendocrine carcinoma.

Primary cutaneous neuroendocrine carcinoma, or Merkel cell carcinoma (MCC), cannot be distinguished morphologically from small-cell neuroendocrine carcinomas (SmCC) from other sites. Immunohistochemistry is required to confirm cutaneous origin, and is also used for detection of sentinel lymph node (SLN) metastases of MCC. Cytokeratin 20 (CK20) expression is commonly used for these purposes, but is negative in some MCC cases, and has unclear specificity. We evaluated immunohistochemistry for neurofilament and CK20 in MCC compared with SmCC from other sites. We evaluated neurofilament expression in 55 MCC specimens from 39 unique patients, including nine CK20-negative MCC tumours. Neurofilament expression was observed in 42 of 55 (76.4%) MCC cases, including seven of nine (77.8%) CK20-negative MCC cases. Neurofilament was expressed in nine of 12 (75%) Merkel cell polyomavirus-positive tumours and five of 10 (50%) virus-negative tumours. Compared to a standard immunohistochemical panel (cytokeratin cocktail and CK20), neurofilament was 87.5% sensitive for detecting SLN metastases. Neurofilament and CK20 expression was also assessed in 61 extracutaneous SmCC from 60 unique patients, with primary sites including lung (27), bladder (18), cervix (3), gastrointestinal tract (3), sinonasal tract (2) and other sites (7). The specificity of neurofilament and CK20 for MCC versus non-cutaneous SmCC was 96.7% and 59.0%, respectively. Neurofilament has superior specificity to CK20 in distinguishing MCC from non-cutaneous SmCC. Neurofilament is frequently expressed in CK20- and virus-negative MCC tumours. Limitations of neurofilament immunohistochemistry include lower sensitivity than CK20 and subtle staining in some tumours. However, our findings indicate that neurofilament is useful for excluding non-cutaneous SmCC.

Therapeutic effect of perinatal exogenous melatonin on behavioral and histopathological changes and antioxidative enzymes in neonate mouse model of cortical malformation

BackgroundMelatonin, which is an antioxidant and neuroprotective agent, can be an effective treatment for neurological disorders. We assessed the effect of melatonin administration on histological changes, antioxidant enzyme levels, and behavioral changes in a neonate mouse model of cortical malformation. Materials and methodsCortical malformation was induced by two injections of 15 mg/kg methylazoxymethanol (MAM) on gestational day 15 (E15). Pregnant Balb/c mice were randomly divided into the following six groups: Control (CO), Melatonin (MEL), Luzindole (LUZ), MAM, MEL + MAM1 (co-treatment), and MEL + MAM2 (pretreatment). Melatonin was intraperitoneally injected at a dose of 10 mg/kg daily (from E15 until delivery of from E6 for 20 days after delivery). On postnatal day 31, the activity and anxiety of mice were assessed by open field and elevated plus maze tests, respectively. Histopathological changes in the neonate cortex were studied using hematoxylin and eosin staining and neurofilament immunohistochemistry. Enzyme-linked immunosorbent assays were used to measure the activity of nitric oxide (NO), malondialdehyde (MDA), and antioxidant enzymes, including catalase (CAT), super oxide dismutase (SOD), and glutathione peroxidase (GPX). ResultsIn the behavioral assessment of neonate mice, a significant increase in the crossing activity and decrease in anxiety were recorded in groups treated with MAM plus melatonin. In histological examination, heterotopic, dysmorphic, and ectopic cells, as well as dyslamination, were seen in the MAM and LUZ groups. However, these defects were attenuated in the MAM plus melatonin groups. Significant reductions were recorded in the SOD and GPX levels in the MAM and LUZ groups compared to the control, while the NO level was increased in these groups. Groups that received MAM plus melatonin showed significant increases in the levels of SOD and GPX and a significant decrease in the level of NO, compared to the MAM group. ConclusionMelatonin increased the crossing activity and decreased the anxiety in the treated mice of the neonate mouse model of cortical malformation. Histologically, the administration of exogenous melatonin in pregnant mice and their neonates had a protective effect on the cerebral cortex of neonates. Also, this effect is elicited by decreasing NO and increasing antioxidative enzymes.

347 Histopathology of Necrotic Spinal Cord Tissue Exudate Collected During Surgical Implantation of a Biodegradable Scaffold Following Acute Spinal Cord Injury

Abstract INTRODUCTION Acute implantation of biodegradable scaffolds following spinal cord injury (SCI) has been shown pre-clinically to reduce chronic cavitation, increase white matter sparing, and increase the deposition of neuropermissive remodeled tissue. The surgical procedure of scaffold implantation allows for the gentle removal of acutely necrotic tissue resulting in a cavity in which the scaffold is placed. Here we report for the first time on the histopathological findings in both animal and human tissue specimens. METHODS Pre-clinically, experimental spinal cord contusion injuries were performed as previously reported in pigs. Clinically, the ongoing INSPIRE study (NCT02138110) is currently enrolling baseline T2-T12/L1 subjects with neurologically complete (AIS A) SCI within 96 hours of injury. The surgical procedure for implantation consists of durotomy and sometimes myelotomy. Commonly, damaged spinal cord tissue under pressure spontaneously extrudes after piotomy in pure contusion injuries. This tissue sample is collected and submitted for histopathologic analysis. RESULTS &gt;24 hours post severe contusion/compression injury in a pig model, hematoxylin and eosin-stained (H&amp;E) paraffin sections revealed myelin and axonal degeneration along with numerous scattered spheroids (swollen axons) with hemorrhage and acute inflammation at the wound site. Surgical pathology reports document neuropil disruption and devitalization in samples collected during surgery at 40 and 82 hours post-injury. In the patient implanted at 40 hours, the tissue specimen contained fragments of disrupted neuropil with swollen and fragmented axons as evaluated by H&amp;E and neurofilament immunohistochemistry. CONCLUSION Severe SCI leads to the rapid formation of irreversibly damaged parenchyma. Our findings in animal and human tissue samples revealed acute tissue disruption and devitalization within 24–82 hours post-injury. This time frame was too short to appreciate phagocytosis, gliosis, or axon sprouts. Future patient enrollment and tissue collection in the ongoing clinical study will continue to build upon these initial observations.

Neurons of the rat cervical spinal cord express vimentin and neurofilament after intraparenchymal injection of kainic acid

Intermediate filaments (IF) can be altered under disorders such as neurodegenerative diseases. Kainic acid (KA) induce behavioral changes and histopathological alterations of the spinal cord of injected rats. Our goal was to evaluate the IF expression in neurons during this injury model. Animals were injected with KA at the C5 segment of the cervical spinal cord and euthanized at 1, 3 and 7 post injection (pi) days. Neuronal cell counting showed a significant loss of neurons at the injection site when compared with those of sham and non-operated animals. Immunohistochemistry for vimentin and neurofilament showed positive labeling of perikarya in sham and KA-injected animals since day 1 pi that lasted for the remaining experimental days. Colocalization analysis between enolase and vimentin or neurofilament confirmed a high index of colocalization in both experimental groups at day 1 pi. This index decreased in sham animals by day 3 pi whereas that of KA-injected animals remained high throughout the experiment. These results may suggest that perikarya initiate an unconventional IF expression, which may respond to the neuronal damage induced by the mechanical injury and the excitotoxic effect of KA. It seems that vimentin and neurofilament expression may be a necessary change to promote recovery of the damaged tissue.

Pathological Confirmation of Optic Neuropathy in Familial Dysautonomia

Clinical data suggest that optic neuropathy and retinal ganglion cell loss are the main cause of visual decline in patients with familial dysautonomia, but this has not previously been confirmed by pathological analyses. We studied retinas and optic nerves in 6 eyes from 3 affected patients obtained at autopsy. Analyses included routine neurohistology and immunohistochemistry for neurofilaments, cytochrome c oxidase (COX), and melanopsin-containing ganglion cells. We observed profound axon loss in the temporal portions of optic nerves with relative preservation in the nasal portions; this correlated with clinical and optical coherence tomography findings in 1 patient. Retinal ganglion cell layers were markedly reduced in the central retina, whereas melanopsin-containing ganglion cells were relatively spared. COX staining was reduced in the temporal portions of the optic nerve indicating reduced mitochondrial density. Axonal swelling with degenerating lysosomes and mitochondria were observed by electron microscopy. These findings support the concept that there is a specific optic neuropathy and retinopathy in patients with familial dysautonomia similar to that seen in other optic neuropathies with mitochondrial dysfunction. This raises the possibility that defective expression of the IkB kinase complex-associated protein (IKAP) resulting from mutations in IKBKAP affects mitochondrial function in the metabolism-dependent retinal parvocellular ganglion cells in this condition.

Regional Myelin and Axon Damage and Neuroinflammation in the Adult Mouse Brain After Long-Term Postnatal Vanadium Exposure.

Environmental exposure to vanadium occurs in areas of persistent burning of fossil fuels; this metal is known to induce oxidative stress and oligodendrocyte damage. Here, we determined whether vanadium exposure (3 mg/kg) in mice during the first 3 postnatal months leads to a sustained neuroinflammatory response. Body weight monitoring, and muscle strength and open field tests showed reduction of body weight gain and locomotor impairment in vanadium-exposed mice. Myelin histochemistry and immunohistochemistry for astrocytes, microglia, and nonphosphorylated neurofilaments revealed striking regional heterogeneity. Myelin damage involved the midline corpus callosum and fibers in cortical gray matter, hippocampus, and diencephalon that were associated with axonal damage. Astrocyte and microglial activation was identified in the same regions and in the internal capsule; however, no overt myelin and axon damage was observed in the latter. Double immunofluorescence revealed induction of high tumor necrosis factor (TNF) immunoreactivity in reactive astrocytes. Western blotting analysis showed significant induction of TNF and interleukin-1β expression. Together these findings show that chronic postnatal vanadium exposure leads to functional deficit and region-dependent myelin damage that does not spare axons. This injury is associated with glial cell activation and proinflammatory cytokine induction, which may reflect both neurotoxic and neuroprotective responses.

Alteration of microRNA expressions in the pons and medulla in rats after 3,3'-iminodipropionitrile administration.

Although 3,3′-iminodipropionitrile (IDPN) is widely used as a neurotoxicant to cause axonopathy due to accumulation of neurofilaments in several rodent models, its mechanism of neurotoxicity has not been fully understood. In particular, no information regarding microRNA (miRNA) alteration associated with IDPN is available. This study was conducted to reveal miRNA alteration related to IDPN-induced neurotoxicity. Rats were administered IDPN (20, 50, or 125 mg/kg/day) orally for 3, 7, and 14 days. Histopathological features were investigated using immunohistochemistry for neurofilaments and glial cells, and miRNA alterations were analyzed by microarray and reverse transcription polymerase chain reaction. Nervous symptoms such as ataxic gait and head bobbing were observed from Day 9 at 125 mg/kg. Axonal swelling due to accumulation of neurofilaments was observed especially in the pons, medulla, and spinal cord on Day 7 at 125 mg/kg and on Day 14 at 50 and 125 mg/kg. Furthermore, significant upregulation of miR-547* was observed in the pons and medulla in treated animals only on Day 14 at 125 mg/kg. This is the first report indicating that miR-547* is associated with IDPN-induced neurotoxicity, especially in an advanced stage of axonopathy.

Topography and extent of pulmonary vagus nerve supply with respect to transthoracic oesophagectomy.

Pulmonary complications are frequently observed after transthoracic oesophagectomy. These complications may be reduced by sparing the vagus nerve branches to the lung. However, current descriptions of the regional anatomy are insufficient. Therefore, we aimed to provide a highly detailed description of the course of the pulmonary vagus nerve branches. In six fixed adult human cadavers, bilateral microscopic dissection of the vagus nerve branches to the lungs was performed. The level of branching and the number, calibre and distribution of nerve branches were described. Nerve fibres were identified using neurofilament immunohistochemistry, and the nerve calibre was measured using computerized image analysis. Both lungs were supplied by a predominant posterior and a smaller anterior nerve plexus. The right lung was supplied by 13 (10-18) posterior and 3 (2-3) anterior branches containing 77% (62-100%) and 23% (0-38%) of the lung nerve supply, respectively. The left lung was supplied by a median of 12 (8-13) posterior and 3 (2-4) anterior branches containing 74% (60-84%) and 26% (16-40%) of the left lung nerve supply, respectively. During transthoracic oesophagectomy with en bloc lymphadenectomy and transection of the vagus nerves at the level of the azygos vein, 68-100% of the right lung nerve supply and 86-100% of the inferior left lung lobe nerve supply were severed. When vagotomy was performed distally to the last large pulmonary branch, 0-8% and 0-13% of the nerve branches to the right middle/inferior lobes and left inferior lobe, respectively, were lost. In conclusion, this study provides a detailed description of the extensive pulmonary nerve supply provided by the vagus nerves. During oesophagectomy, extensive mediastinal lymphadenectomy denervates the lung to a great extent; however, this can be prevented by performing the vagotomy distal to the caudalmost large pulmonary branch. Further research is required to determine the feasibility of sparing the pulmonary vagus nerve branches without compromising the completeness of lymphadenectomy.

Xenotransplantation of human adipose-derived stem cells in the regeneration of a rabbit peripheral nerve

To promote end-to-end nerve fiber contacts of a 4-cm gap in the peroneal nerve of white New Zealand rabbits, an autologous vein conduit was used and three groups of animals were evaluated. In Group I, the gap was repaired with a vein conduit refilled with fibrin. Group II was similar, but the animals were treated with cyclosporine A. In Group III, a fibrin scaffold with hAdMSCs was placed inside the autologous vein conduit, and animals were treated with cyclosporine A. Neurofilament immunohistochemistry results showed 100% nerve regeneration at the vein guidance channel 90 days after the surgery in the hAdMSC-transplanted group but lesser neural regeneration in the neurofilaments of groups I and II. The analysis of variance (ANOVA) test showed statistically significant differences among all groups (p<0.04). Group III exclusively tested positive for human monoclonal anti-mitochondrial antibody. Electron microscopy images showed tiny bundles, with a predominance of nonmyelinated axons. Myelinated axons caused irregular thickness of the myelin sheath, which was especially observed in group III. Xenotransplantation of hAdMSCs into a fibrin scaffold promoted nerve regeneration through a vein conduit that connected a 4-cm gap created at the peroneal nerve of rabbits. Animals treated with hAdMSCs presented negative inflammatory response at the regenerated nerve gaps, but it was demonstrated that hAdMSCs were incorporated to the new nerve creating neural tissue and endothelial cells. However, hAdMSCs required immunosuppression with cyclosporine A to achieve axonal regeneration.

Muscle reinnervation with nerve-muscle-endplate band grafting technique: correlation between force recovery and axonal regeneration

BackgroundThis study was designed to determine the correlation between functional recovery and the extent of axonal regeneration after muscle reinnervation with our recently developed nerve-muscle-endplate band grafting (NMEG) technique in a rat model. Materials and methodsThe right experimentally paralyzed sternomastoid (SM) muscle by nerve transection was immediately reinnervated with an NMEG pedicle harvested from a neighboring sternohyoid muscle. The NMEG pedicle contained a muscle block (6 × 6 × 3 mm), a donor nerve branch with nerve terminals, and a motor endplate band. Three months after surgery, the tetanic force of the SM muscle was measured and the regenerated axons in the muscle were detected using neurofilament immunohistochemistry. ResultsThe results showed that the maximal tetanic force (a measure of muscle functional recovery) of the NMEG-reinnervated SM muscle reached up to 66.0% of the normal control. The wet weight of the reinnervated SM muscle (a measure of muscle mass recovery) was 87.2% of the control. The area fraction of the regenerating axons visualized with neurofilament staining within the NMEG-reinnervated SM muscle (a measure of muscle reinnervation) was 42.3%. A positive correlation was revealed between the extent of muscle reinnervation and maximal muscle force. ConclusionsOur newly developed NMEG technique results in satisfactory functional outcomes and nerve regeneration. Further improvement in the functional recovery after NMEG reinnervation could be achieved by refining the surgical procedure and creating an ideal environment that favors axon-endplate connections and accelerates axonal growth and sprouting.

Dexmedetomidine, α2-adrenoceptor agonist, does not induce apoptosis in the brachial plexus of rats

Dexmedetomidine (Precedex) is a selective α2-adrenoceptor agonist that shows opioid-sparing effects with reduced inhalational anesthetic requirement. Since the possibility that dexmedetomidine could induce nerve degeneration has not been completely ruled out, we evaluated whether it can induce apoptosis in rats when used in a nerve block. We performed hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays, immunohistochemistry for caspase-3 and neurofilament, and western blotting for BAX, Bcl-2, and nerve growth factor (NGF) in the brachial plexus of rats. The axillary brachial plexus was carefully exposed and 100 μl (40 µg/kg) dexmedetomidine was administered directly. Rats were sacrificed at 6 h, 12 h, or 24 h after dexmedetomidine administration. In the present study, dexmedetomidine did not exert any effects on histological findings and neurofilament expression. The number of TUNEL-positive and caspase-3-positive cells in the brachial plexus did not change following dexmedetomidine administration. The Bcl-2 to BAX ratio temporarily increased and then returned to the control level at 24 h after dexmedetomidine administration. Expression of NGF in the brachial plexus temporarily increased and then returned to the control level 12 h after dexmedetomidine administration. The results of our study demonstrated that dexmedetomidine did not induce apoptosis and degeneration when used in the brachial plexus.

Enhanced immunohistochemical detection of neural infiltration in primary melanoma: is there a clinical value?

Neural infiltration in primary melanoma is a histopathologic feature that has been associated with desmoplastic histopathologic subtype and local recurrence in the literature. We tested the hypothesis that improved detection and characterization of neural infiltration into peritumoral or intratumoral location and perineural or intraneural involvement could have a prognostic relevance. We studied 128 primary melanoma cases prospectively accrued and followed at New York University using immunohistochemical detection with antihuman neurofilament protein and routine histology with hematoxylin and eosin. Neural infiltration, defined as the presence of tumor cells involving or immediately surrounding nerve foci, was identified and characterized using both detection methods. Neural infiltration rate of detection was enhanced by immunohistochemistry for neurofilament in matched-pair design (47% by immunohistochemistry versus 25% by routine histology). Immunohistochemical detection of neural infiltration was significantly associated with ulceration (P = .021), desmoplastic and acral lentiginous histologic subtype (P = .008), and head/neck/hands/feet tumor location (P = .037). Routinely detected neural infiltration was significantly associated with local recurrence (P = .010). Immunohistochemistry detected more intratumoral neural infiltration cases compared with routine histology (30% versus 3%, respectively). Peritumoral and intratumoral nerve location had no impact on clinical outcomes. Using a multivariate model controlling for stage, neither routinely detected neural infiltration nor enhanced immunohistochemical characterization of neural infiltration was significantly associated with disease-free or overall survival. Our data demonstrate that routinely detected neural infiltration is associated with local recurrence in all histologic subtypes but that improved detection and characterization of neural infiltration with immunohistochemistry in primary melanoma does not add to prognostic relevance.

Is the denervation or hyperinnervation of the cardiac sympathetic nerve in the subepicardium related to unexpected cardiac death?

Past studies have reported that abnormal innervation of cardiac sympathetic nerve can cause sudden cardiac death through the arrythmogenesis; however, the severe cardiac sympathetic degeneration does not necessarily cause clinical problems. This study aimed to examine whether denervation or hyperinnervation of cardiac sympathetic nerves in the subepicardium is associated with unexpected cardiac death (UCD). Cardiac tissues of 278 forensic autopsy cases within 48 h after death were analyzed by double-staining immunohistochemistry for tyrosine hydroxylase and neurofilament. The density of nerve fascicles and the degeneration rate in the subepicardium of the left ventricular anterior wall were compared between the UCD group and the non-UCD group. The density of nerve fascicles was lower in the SCD group (median: 51.9/cm(2)) than in the non-SCD group (median: 58.9/cm(2)); however, the difference was not significant (P = .08). The degeneration rate was higher in the SCD group (median: 0.19) than in the non-SCD group (median: 0.17), but again, the difference was not significant (P = .43). The multiple logistic regression model did not show a significant association between the incidence of UCD and the density of nerve fascicles or the degeneration rate. It cannot be concluded that the denervation or hyperinnervation of cardiac sympathetic nerves in the subepicardium is related to UCD. Abnormal innervation of cardiac sympathetic nerves in the subepicardium may not have a substantial effect on UCD, compared to other arrhythmogenic factors.

Age-related changes in the brain of obese Zucker rat:Morphological and immunochemical analysis.

Metabolic syndrome (MetS) is defined as a combination of glucose intolerance, arterial hypertension, dyslipidemia and obesity with insulin resistance as the main cause of these imbalances. In general, obesity increases the risk of vascular diseases. Both cardiovascular and cerebrovascular disorders are often linked to impaired cognitive functions. A relationship between metabolic disease and mild cognitive impairment, and/or vascular dementia was hypothesized. The obese Zucker rat (OZR), represents a model of obesity and type 2 diabetes exhibiting a moderate degree of arterial hypertension. OZRs are characterized by the simultaneous occurrence of hyperglycaemia, hyperinsulinaemia and hyperlipidaemia. This study has investigated brain microanatomy of OZRs compared with their nonobese cohort lean Zucker rats (LZRs) to assess possible relationships between MetS and brain damage. Male OZRs and LZRs of 12, 16 and 20 weeks of age were used. Body weight, blood pressure and blood chemistry parameters were checked every two weeks and before killing. The brain was dissected out and processed for immunochemical and immunohistochemical analysis of nerve cells identified by neuronal specific nuclear protein (NeuN) and axons identified by neurofilament (NF) immunohistochemistry. Glial-fibrillary acid protein (GFAP) immunoreactive astrocytes were also investigated. OZRs of different ages, were characterized by a high body weight, an increase of systolic pressure, glycemia, triglycerides and cholesterol levels in comparison with LZRs. An age-dependent increase of these parameters was observed in OZRs. A decrease of brain/body weight ratio was found in OZRs. In frontal cortex and hippocampus, morphological and immunochemical analysis revealed a decrease of NeuN immunoreactive neurons not related to apoptosis in older OZRs group compared to age matched LZRs. In OZRs a decrease of NF immunoreaction and an increase of GFAP immunoreactive astrocytes was observed compared to LZRs These findings suggest that OZRs, used for investigating mechanisms and pathophysiology of obesity and type 2 diabetes, may also represent a model for assessing the influence of MetS on brain.