Neurofilament Immunoreactivity Research Articles

Neuronal Distribution in Colorectal Cancer: Associations With Clinicopathological Parameters and Survival

Over the past years, insights in the cancer neuroscience field increased rapidly, and a potential role for neurons in colorectal carcinogenesis has been recognized. However, knowledge on the neuronal distribution, subtypes, origin, and associations with clinicopathological characteristics in human studies is sparse. In this study, colorectal tumor tissues from the Netherlands Cohort Study on diet and cancer (n = 490) and an in-cohort validation population (n = 529) were immunohistochemically stained for the pan-neuronal markers neurofilament (NF) and protein gene product 9.5 (PGP9.5) to study the association between neuronal marker expression and clinicopathological characteristics. In addition, tumor and healthy colon tissues were stained for neuronal subtype markers, and their immunoreactivity in colorectal cancer (CRC) stroma was analyzed. NF-positive and PGP9.5-positive nerve fibers were found within the tumor stroma and mostly characterized by the neuronal subtype markers vasoactive intestinal peptide and neuronal nitric oxide synthase, suggesting that inhibitory neurons are the most prominent neuronal subtype in CRC. NF and PGP9.5 protein expression were not consistently associated with tumor stage, sublocation, differentiation grade, and median survival. NF immunoreactivity was associated with a worse CRC-specific survival in the study cohort (P = .025) independent of other prognostic factors (hazard ratio, 2.31; 95% CI, 1.33-4.03; P = .003), but these results were not observed in the in-cohort validation group. PGP9.5, in contrast, was associated with a worse CRC-specific survival in the in-cohort validation (P = .046) but not in the study population. This effect disappeared in multivariate analyses (hazard ratio, 0.81; 95% CI, 0.50-1.32; P = .393), indicating that this effect was dependent on other prognostic factors. This study demonstrates that the tumor stroma of CRC patients mainly harbors inhibitory neurons and that NF as a single marker is significantly associated with a poorer CRC-specific survival in the study cohort but necessitates future validation.

Axonal Protection by Oral Nicotinamide Riboside Treatment with Upregulated AMPK Phosphorylation in a Rat Glaucomatous Degeneration Model.

Nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD+), has been studied to support human health against metabolic stress, cardiovascular disease, and neurodegenerative disease. In the present study, we investigated the effects of oral NR on axonal damage in a rat ocular hypertension model. Intraocular pressure (IOP) elevation was induced by laser irradiation and then the rats received oral NR of 1000 mg/kg/day daily. IOP elevation was seen 7, 14, and 21 days after laser irradiation compared with the controls. We confirmed that oral NR administration significantly increased NAD+ levels in the retina. After 3-week oral administration of NR, morphometric analysis of optic nerve cross-sections showed that the number of axons was protected compared with that in the untreated ocular hypertension group. Oral NR administration significantly prevented retinal ganglion cell (RGC) fiber loss in retinal flat mounts, as shown by neurofilament immunostaining. Immunoblotting samples from the optic nerves showed that oral NR administration augmented the phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) level in rats with and without ocular hypertension induction. Immunohistochemical analysis showed that some p-AMPK-immunopositive fibers were colocalized with neurofilament immunoreactivity in the control group, and oral NR administration enhanced p-AMPK immunopositivity. Our findings suggest that oral NR administration protects against glaucomatous RGC axonal degeneration with the possible upregulation of p-AMPK.

Neurofilament light chain is increased in the parahippocampal cortex and associates with pathological hallmarks in Parkinson’s disease dementia

BackgroundIncreased neurofilament levels in biofluids are commonly used as a proxy for neurodegeneration in several neurodegenerative disorders. In this study, we aimed to investigate the distribution of neurofilaments in the cerebral cortex of Parkinson’s disease (PD), PD with dementia (PDD) and dementia with Lewy bodies (DLB) donors, and its association with pathology load and MRI measures of atrophy and diffusivity.MethodsUsing a within-subject post-mortem MRI-pathology approach, we included 9 PD, 12 PDD/DLB and 18 age-matched control donors. Cortical thickness and mean diffusivity (MD) metrics were extracted respectively from 3DT1 and DTI at 3T in-situ MRI. After autopsy, pathological hallmarks (pSer129-αSyn, p-tau and amyloid-β load) together with neurofilament light-chain (NfL) and phosphorylated-neurofilament medium- and heavy-chain (p-NfM/H) immunoreactivity were quantified in seven cortical regions, and studied in detail with confocal-laser scanning microscopy. The correlations between MRI and pathological measures were studied using linear mixed models.ResultsCompared to controls, p-NfM/H immunoreactivity was increased in all cortical regions in PD and PDD/DLB, whereas NfL immunoreactivity was increased in the parahippocampal and entorhinal cortex in PDD/DLB. NfL-positive neurons showed degenerative morphological features and axonal fragmentation. The increased p-NfM/H correlated with p-tau load, and NfL correlated with pSer129-αSyn but more strongly with p-tau load in PDD/DLB. Lastly, neurofilament immunoreactivity correlated with cortical thinning in PD and with increased cortical MD in PDD/DLB.ConclusionsTaken together, increased neurofilament immunoreactivity suggests underlying axonal injury and neurofilament accumulation in morphologically altered neurons with increased pathological burden. Importantly, we demonstrate that such neurofilament markers at least partly explain MRI measures that are associated with the neurodegenerative process.

Protective effect of short-term thymoquinone administration on the central nervous system in cisplatin-induced neurotoxicity.

This study was performed to investigate the potential beneficial effects of thymoquinone (TQ) on brain tissue based on biochemical and histopathological analyses in cisplatin (CIS) treated rats with central nervous system (CNS) neurotoxicity. The rats were randomly divided into 4 groups with 8 rats in each group (n:8). Group 1: (Control), saline was administered for 3 days at a volume of 0.5 ml per day intraperitoneal (i.p.). Group 2: (CIS Group), one dose of CIS was administered (7 mg/kg i.p.). Group 3: (TQ Group), TQ was given at a dose of 5 mg/kg per day for 3 days (i.p.). Group 4: (CIS+TQ Group), one dose of 7 mg/kg was initiated half an hour before administration of CIS and one dose of 5 mg/kg per day was administered TQ i.p. for 3 days. Malondialdehyde levels were found to be statistically significantly higher in the CIS group compared to the control group. Degenerative changes observed in the CIS+TQ group were found to be milder than in the CIS group. In the CIS+TQ group, a statistically significant decrease in the severity of caspase-3 immunoreactivity was found when compared to the CIS group. It was found that the severity of neurofilament immunoreactivity monitored in neuronal extensions was similar in all groups. In the CIS+TQ group, the severity of tau protein's immunoreactivity was similar to that of the CIS-group. According to the results obtained in our study, beneficial effects were obtained in reducing neurotoxicity with short-term TQ application in rats treated with CIS treatment.

Activity in nodose ganglia neurons after treatment with CP 55,940 and cholecystokinin.

Previous work has shown that cannabinoids increase feeding, while cholecystokinin (CCK) has an anorexigenic effect on food intake. Receptors for these hormones are located on cell bodies of vagal afferent nerves in the nodose ganglia. An interaction between CCK and endocannabinoid receptors has been suggested. The purpose of these studies is to explore the effect of pretreatment with a cannabinoid agonist, CP 55,940, on nodose neuron activation by CCK. To determine the effect of CP 55,940 and CCK on neuron activation, rats were anesthetized and nodose ganglia were excised. The neurons were dissociated and placed in culture on coverslips. The cells were treated with media; CP 55,940; CCK; CP 55,940 followed by CCK; or AM 251, a CB1 receptor antagonist, and CP 55,940 followed by CCK. Immunohistochemistry was performed to stain the cells for cFos as a measure of cell activation. Neurons were identified using neurofilament immunoreactivity. The neurons on each slip were counted using fluorescence imaging, and the number of neurons that were cFos positive was counted in order to calculate the percentage of activated neurons per coverslip. Pretreatment with CP 55,940 decreased the percentage of neurons expressing cFos‐immunoreactivity in response to CCK. This observation suggests that cannabinoids inhibit CCK activation of nodose ganglion neurons.

Grading Gangliogliomas: a Short Case Series With Clinico-Imagistic and Immunohistopathological Correlations.

Ganglioglioma (GG) represents an extremely rare tumor of the central nervous system, which is composed of two different cellular populations: a glial cell population and a neuronal cell population, the former being the one which will establish the histologic grade of the tumor. The current World Health Organization (WHO) Classification of Tumors of the Central Nervous System divides gangliogliomas into benign (WHO grade I) and malignant (WHO grade III). Several scientific studies acknowledge that some tumors are difficult to grade but, due to the scarcity of cases as well as the lack of multicentric epidemiological data, there are no extensive studies regarding this matter in the neuropathology literature. We report a short case series of three patients with ganglioglioma who were admitted and treated at the Neurosurgery Department of "Bagdasar Arseni" Emergency Hospital. The patients had different clinical presentations, varying from migraines and epileptic seizures to development of a large, slowly growing tumor. Tissue fragments were obtained through surgical resection and sent to the Pathology Department for microscopic investigation. Histopathologic examination revealed both components of the tumor, supporting the diagnosis of ganglioglioma, albeit the glial component featured different histologic grade in each tumor. The tumor diagnosed as grade II lacked mitoses, but showed conspicuous atypia and numerous multinucleated cells. Immunohistochemistry revealed immunoreactivity for synaptophysin, chromogranin A and neurofilament in the neuronal component and GFAP positivity in the glial component of the tumor. Neurofilament showed an unusual pattern of staining, in which areas with benign features showed patchy positivity, while areas with malignant features and striking nuclear pleomorphism were completely negative. Due to the completely different clinical outcome, we strongly believe that a grade II ganglioglioma should be differentiated from a grade III GG, based on the lack of mitoses, necrosis and microvascular proliferation. The differentiation between grade II GG and grade I GG should be made on the cellular pleomorphism of both components (glial and neuronal). Based on our experience, we conclude that immunohistochemistry could aid in this differentiation through markers like: Ki67, neurofilament, CD34 and chromogranin A. We strongly believe that further immunohistochemical research on larger study groups will eventually lead to a consensus regarding definitive criteria for grade II gangliogliomas.

Treatment with acetyl-L-carnitine exerts a neuroprotective effect in the sciatic nerve following loose ligation: a functional and microanatomical study.

Peripheral neuropathies are chronic painful syndromes characterized by allodynia, hyperalgesia and altered nerve functionality. Nerve tissue degeneration represents the microanatomical correlate of peripheral neuropathies. Aimed to improve the therapeutic possibilities, this study investigated the hypersensitivity and the neuromorphological alterations related to the loose ligation of the sciatic nerve in rats. Effects elicited by treatment with acetyl-L-carnitine (ALCAR) in comparison to gabapentin were assessed. Axonal injury, reduction of myelin deposition and accumulation of inflammatory cells were detected in damaged nerve. A decrease of phosphorylated 200-kDa neurofilament (NFP) immunoreactivity and a redistribution in small clusters of myelin basic like-protein (MBP) were observed in ipsilateral nerves. Treatment with ALCAR (100 mg/kg intraperitoneally - i.p.) and gabapentin (70 mg/kg i.p.) administered bis in die for 14 days induced a significant pain relieving effect. ALCAR, but not gabapentin, significantly countered neuromorphological changes and increased axonal NFP immunoreactivity. These findings indicate that both ALCAR and gabapentin significantly decreased the hypersensitivity related to neuropathic lesions. The observation of the positive ALCAR effect on axonal and myelin sheath alterations in damaged nerve supports its use as neurorestorative agent against neuropathies through mechanism(s) consistent to those focused in this study.

Therapeutic benefit of environmental enrichment on optic neuritis

Optic neuritis (ON) is an inflammatory, demyelinating, neurodegenerative, and presently untreatable condition of the optic nerve which might induce blindness. We analyzed the effect of environmental enrichment (EE) on visual pathway damage provoked by experimental ON induced by a microinjection of bacterial lipopolysaccharide (LPS) into the optic nerve. For this purpose, LPS was microinjected into the optic nerve from male Wistar rats. After injection, one group of animals was submitted to EE, and another group remained in standard environment (SE) for 21 days. EE prevented the decrease in pupil light reflex (PLR), visual evoked potentials, retinal anterograde transport, phosphorylated neurofilament immunoreactivity, myelination (luxol fast blue staining), and axon (toluidine blue staining) and retinal ganglion cell (Brn3a-immunoreactivity) number. EE also prevented microglial/macrophage reactivity (Iba-1- and ED1-immunoreactivity), and astrocytosis (glial fibrillary acidic protein-immunostaining) induced by experimental ON. LPS-injected optic nerves displayed oxidative damage and increased inducible nitric oxide synthase, cyclooxygenase-2, and interleukin-1β and TNFα mRNA levels which were prevented by EE. EE increased optic nerve brain-derived neurotrophic factor levels. When EE started at 4 (but not 7) days post-injection of LPS, a preservation of the PLR was observed at 21 days post-LPS, which was blocked by the daily administration of ANA-12 from day 4 to day 7 post-LPS. Moreover, EE from day 4 to day 7 post-LPS significantly preserved the PLR at 21 days post-injection. Taken together, our data suggest that EE preserved visual functions and reduced neuroinflammation of the optic nerve.This article is part of the Special Issue entitled “Neurobiology of Environmental Enrichment”.

Landolphia owariensis Attenuates Alcohol-induced Cerebellar Neurodegeneration: Significance of Neurofilament Protein Alteration in the Purkinje Cells.

Alcohol-induced cerebellar neurodegeneration is a neuroadaptation that is associated with chronic alcohol abuse. Conventional drugs have been largely unsatisfactory in preventing neurodegeneration. Yet, multimodal neuro-protective therapeutic agents have been hypothesised to have high therapeutic potential for the treatment of CNS conditions; there is yet a dilemma of how this would be achieved. Contrarily, medicinal botanicals are naturally multimodal in their mechanism of action. The effect of L. owariensis was therefore assessed in alcohol-induced neurodegeneration of the cerebellar cortex in rats. Two groups of rats were oro-gastrically fed thrice daily with 5 g/kg ethanol (25% w/v), and 5 g/kg ethanol (25% w/v) plus L. owariensis (100 mg/kg body weight) respectively in diluted nutritionally complete diet (50% v/v). A control group was correspondingly fed a nutritionally complete diet (50% v/v) made isocaloric with glucose. Cytoarchitectural study of the cerebellar cortex was examined with H&E. Immunocytochemical analysis was carried out with the use of monoclonal antibody anti-NF in order to detect alterations in the neuronal cytoskeleton. After 4 days of binge alcohol treatment, we observed that L. owariensis supplementation significantly lowered the levels of histologic and biochemical indices of neurodegeneration. The level of neurodegeneration and cytoarchitecture distortion of the cerebellar cortex of rats exposed to ethanol was reduced by L. owariensis. Neurofilament-immunoreactivity (NF-IR) was evoked in the Purkinje cells of rats that received L. owariensis supplement. L. owariensis attenuates alcohol-induced cerebellar degeneration in the rat by alleviating oxidative stress and alteration of NF protein expression in the Purkinje cells.

Characterization of novel dystonia musculorum mutant mice: Implications for central nervous system abnormality

We identified a novel spontaneous mutant mouse showing motor symptoms that are similar to those of the dystonia musculorum (dt) mouse. The observations suggested that the mutant mice inherited the mild dt phenotype as an autosomal recessive trait. Linkage analysis showed that the causative gene was located near D1Mit373 and D1Mit410 microsatellite markers on chromosome 1, which are close to the dystonin (Dst) gene locus. To investigate whether Dst is the causative gene of the novel mutant phenotype, we crossed the mutant with Dst gene trap (DstGt) mice. Compound heterozygotes showed a typical dt phenotype with sensory degeneration and progressive motor symptoms. DNA sequencing analysis identified a nonsense mutation within the spectrin repeats of the plakin domain. The novel mutant allele was named dt23Rbrc. Motor abnormalities in homozygous dt23Rbrc/dt23Rbrc mice are not as severe as homozygous DstGt/DstGt mice. Histological analyses showed abnormal neurofilament (NF) accumulation in the nervous system of homozygous dt23Rbrc/dt23Rbrc mice, which is characteristic of the dt phenotype. We mapped the distribution of abnormal NF-accumulated neurons in the brain and found that they were located specifically in the brainstem, spinal cord, and in regions such as the vestibular nucleus, reticular nucleus, and red nucleus, which are implicated in posture and motor coordination pathways. The quantification of abnormal NF accumulation in the cytoplasm and spheroids (axons) of neurons showed that abnormal NF immunoreactivity was lower in homozygous dt23Rbrc/dt23Rbrc mice than in homozygous DstGt/DstGt mice. Therefore, we have identified a novel hypomorphic allele of dt, which causes histological abnormalities in the central nervous system that may account for the abnormal motor phenotype. This novel spontaneously occurring mutant may become a good model of hereditary sensory and autonomic neuropathy type 6, which is caused by mutations in the human DST gene.

Immunohistochemical and transcriptome analyses indicate complex breakdown of axonal transport mechanisms in canine distemper leukoencephalitis.

Introduction CDV‐DL (Canine distemper virus‐induced demyelinating leukoencephalitis) represents a spontaneously occurring animal model for demyelinating disorders. Axonopathy represents a key pathomechanism in this disease; however, its underlying pathogenesis has not been addressed in detail so far. This study aimed at the characterization of axonal cytoskeletal, transport, and potential regenerative changes with a parallel focus upon Schwann cell remyelination.MethodsImmunohistochemistry of canine cerebellar tissue as well as a comparative analysis of genes from an independent microarray study were performed.ResultsIncreased axonal immunoreactivity for nonphosphorylated neurofilament was followed by loss of cytoskeletal and motor proteins. Interestingly, a subset of genes encoding for neurofilament subunits and motor proteins was up‐regulated in the chronic stage compared to dogs with subacute CDV‐DL. However, immunohistochemically, hints for axonal regeneration were restricted to up‐regulated axonal positivity of hypoxia‐inducible factor 1 alpha, while growth‐associated protein 43, erythropoietin and its receptor were not or even down‐regulated. Periaxin‐positive structures, indicative of Schwann cell remyelination, were only detected within few advanced lesions.ConclusionsThe present findings demonstrate a complex sequence of axonal cytoskeletal breakdown mechanisms. Moreover, though sparse, this is the first report of Schwann cell remyelination in CDV‐DL. Facilitation of these very limited endogenous regenerative responses represents an important topic for future research.

Arrested development of the dorsal column following neonatal spinal cord injury in the opossum, Monodelphis domestica.

Developmental studies of spinal cord injury in which regrowth of axons occurs across the site of transection rarely distinguish between the recovery of motor-controlling pathways and that of ascending axons carrying sensory information. We describe the morphological changes that occur in the dorsal column (DC) of the grey short-tailed opossum, Monodelphis domestica, following spinal cord injury at two early developmental ages. The spinal cords of opossums that had had their mid-thoracic spinal cords completely transected at postnatal day 7 (P7) or P28 were analysed. Profiles of neurofilament immunoreactivity in transected cords showing DC development were differentially affected by the injury compared with the rest of the cord and cytoarchitecture was modified in an age- and site-dependent manner. The ability of DC neurites to grow across the site of transection was confirmed by injection of fluorescent tracer below the injury. P7 transected cords showed labelling in the DC above the site of original transection indicating that neurites of this sensory tract were able to span the injury. No growth of any neuronal processes was seen after P28 transection. Thus, DC is affected by spinal injury in a differential manner depending on the age at which the transection occurs. This age-differential response, together with other facets of remodelling that occur after neonatal spinal injury, might explain the locomotor adaptations and recovery observed in these animals.

Targeted GAS6 delivery to the CNS protects axons from damage during experimental autoimmune encephalomyelitis.

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNβ injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination.

Rat chronic glaucoma model induced by intracameral injection of microbeads suspended in sodium sulfate–sodium hyaluronate

To establish a rat glaucoma model with chronic intraocular pressure (IOP) elevation induced by microbeads suspended in sodium sulfate-sodium hyaluronate. Chronic elevation of IOP was induced unilaterally by injecting polystyrene microbeads, suspended in 4 % sodium sulfate and 3 % sodium hyaluronate, into the anterior chamber. The microbead suspension was injected through either the clear corneal (CC) or sclerocorneal (SC) tunnel. IOP changes were monitored up to 8 weeks after injection. The loss of retinal ganglion cells (RGCs) was assessed using fluorogold retrograde labeling of RGCs. RGC axons were evaluated by immunohistochemistry and immunoblotting. The resulting IOP elevation was maintained up to 3 weeks after the intracameral injection of microbeads through the CC route and up to 4 weeks after injection through the SC route. The density of RGCs was significantly reduced at 4 weeks after injection, with the SC route leading to more RGC loss than the CC route (p = 0.037). The neurofilament immunoreactivity and protein levels in the optic nerve were also significantly reduced at 4 weeks after injection. Some eyes in the SC route cohort received re-injection of the microbead suspension at 4 weeks after the initial injection, which led to an elevated IOP more than 8 weeks after the initial injection, and eventually a 27.5 % loss of RGC density compared with the control eyes. The intracameral injection of microbeads suspended in hyaluronate effectively produced chronic IOP elevation and subsequent RGC degeneration in rat eyes. The sclerocorneal tunnel approach yielded a longer period of IOP elevation than the clear corneal approach. Our modified microbead injection offers a reliable high-pressure glaucoma model.

Hyperexcitability of Bladder Afferent Neurons Associated with Reduction of Kv1.4 α-Subunit in Rats with Spinal Cord Injury

To clarify the functional and molecular mechanisms inducing hyperexcitability of C-fiber bladder afferent pathways after spinal cord injury we examined changes in the electrophysiological properties of bladder afferent neurons, focusing especially on voltage-gated K channels. Freshly dissociated L6-S1 dorsal root ganglion neurons were prepared from female spinal intact and spinal transected (T9-T10 transection) Sprague Dawley® rats. Whole cell patch clamp recordings were performed on individual bladder afferent neurons. Kv1.2 and Kv1.4 α-subunit expression levels were also evaluated by immunohistochemical and real-time polymerase chain reaction methods. Capsaicin sensitive bladder afferent neurons from spinal transected rats showed increased cell excitability, as evidenced by lower spike activation thresholds and a tonic firing pattern. The peak density of transient A-type K+ currents in capsaicin sensitive bladder afferent neurons from spinal transected rats was significantly less than that from spinal intact rats. Also, the KA current inactivation curve was displaced to more hyperpolarized levels after spinal transection. The protein and mRNA expression of Kv1.4 α-subunits, which can form transient A-type K+ channels, was decreased in bladder afferent neurons after spinal transection. Results indicate that the excitability of capsaicin sensitive C-fiber bladder afferent neurons is increased in association with reductions in transient A-type K+ current density and Kv1.4 α-subunit expression in injured rats. Thus, the Kv1.4 α-subunit could be a molecular target for treating overactive bladder due to neurogenic detrusor overactivity.

A Novel Porcine Model of Traumatic Thoracic Spinal Cord Injury

Spinal cord injury (SCI) researchers have predominately utilized rodents and mice for in vivo SCI modeling and experimentation. From these small animal models have come many insights into the biology of SCI, and a growing number of novel treatments that promote behavioral recovery. It has, however, been difficult to demonstrate the efficacy of such treatments in human clinical trials. A large animal SCI model that is an intermediary between rodent and human SCI may be a valuable translational research resource for pre-clinically evaluating novel therapies, prior to embarking upon lengthy and expensive clinical trials. Here, we describe the development of such a large animal model. A thoracic spinal cord injury at T10/11 was induced in Yucatan miniature pigs (20-25 kg) using a weight drop device. Varying degrees of injury severity were induced by altering the height of the weight drop (5, 10, 20, 30, 40, and 50 cm). Behavioral recovery over 12 weeks was measured using a newly developed Porcine Thoracic Injury Behavior Scale (PTIBS). This scale distinguished locomotor recovery among animals of different injury severities, with strong intra-observer and inter-observer reliability. Histological analysis of the spinal cords 12 weeks post-injury revealed that animals with the more biomechanically severe injuries had less spared white matter and gray matter and less neurofilament immunoreactivity. Additionally, the PTIBS scores correlated strongly with the extent of tissue sparing through the epicenter of injury. This large animal model of SCI may represent a useful intermediary in the testing of novel pharmacological treatments and cell transplantation strategies.

Functional and molecular evidence of myelin‐ and neuroprotection by thyroid hormone administration in experimental allergic encephalomyelitis

Recent data in mouse and rat demyelination models indicate that administration of thyroid hormone (TH) has a positive effect on the demyelination/remyelination balance. As axonal pathology has been recognized as an early neuropathological event in multiple sclerosis, and remyelination is considered a pre-eminent neuroprotective strategy, in this study we investigated whether TH administration improves nerve impulse propagation and protects axons. We followed up the somatosensory evoked potentials (SEPs) in triiodothyronine (T3)-treated and untreated experimental allergic encephalomyelitis (EAE) Dark-Agouti female rats during the electrical stimulation of the tail nerve. T3 treatment started on the 10th day post immunization (DPI) and a pulse administration was continued until the end of the study (33 DPI). SEPs were recorded at baseline (8 DPI) and the day after each hormone/ vehicle administration. T3 treatment was associated with better outcome of clinical and neurophysiological parameters. SEPs latencies of the two groups behaved differently, being briefer and closer to control values (=faster impulse propagation) in T3-treated animals. The effect was evident on 24 DPI. In the same groups of animals, we also investigated axonal proteins, showing that T3 administration normalizes neurofilament immunoreactivity in the fasciculus gracilis and tau hyperphosphorylation in the lumbar spinal cord of EAE animals. No sign of plasma hyperthyroidism was found; moreover, the dysregulation of TH nuclear receptor expression observed in the spinal cord of EAE animals was corrected by T3 treatment. T3 supplementation results in myelin sheath protection, nerve conduction preservation and axon protection in this animal model of multiple sclerosis.

Innervation of cervical ventral facet joint capsule: Histological evidence

To assess the presence of nerves in ventral facet joint capsules as facet capsules are generally implicated in neck pain. Twenty-four ventral cervical facet joint capsules were harvested from 3 unembalmed cadavers. Paraffin sections from these capsules were processed to identify neurofilament and substance P immunoreactive fibers. Nerve fiber presence was also verified by a silver impregnation method. Neurofilament reactive fibers were observed in sections from 9 capsules. They were observed in areas with collagen fibers and areas with irregular connective tissue. Substance P reactive nerve fibers were found in sections from 7 capsules in similar areas. Silver impregnation also revealed the presence of nerve fibers. The nerve fibers were also found as bundles in the lateral margins of the capsule. A Pacinian corpuscle-like ending was also observed in one specimen. Nerve fibers revealed by neurofilament immunoreactivity and silver staining support innervation of the ventral aspect of the facet joint capsule. The presence of substance P reactive fibers supports the potential role of these elements in mediating pain. The presence of a Pacinian-like ending implicates a potential role in joint movement.

Motor nerve terminal destruction and regeneration following anti-ganglioside antibody and complement-mediated injury: An in and ex vivo imaging study in the mouse

Both the neural and glial components of the neuromuscular junction (NMJ) have been identified as potential sites for anti-ganglioside antibody (Ab) binding and complement-mediated injury in murine models for the human peripheral nerve disorder Guillain-Barré syndrome (GBS). Some patients suffering from the acute motor axonal neuropathy (AMAN) forms of GBS recover very rapidly from paralysis; it has been proposed that in these cases the injury was restricted to the distal motor axons and nerve terminals (NTs) which are able to regenerate over a very short time-frame.To test this hypothesis, the ventral neck muscles of mice (n=45) expressing cytosolic fluorescent proteins in their axons (CFP) and Schwann cells (GFP) were subjected to a single topical application of anti-ganglioside Ab followed by a source of complement. Group A (n=15) received Ab that selectively bound to the NTs, group B (n=15) received Abs that bound both to the NTs and the perisynaptic Schwann cells (pSCs) and group C (control animals; n=15) only received complement. Evolution of the injury was documented by in vivo imaging, and following euthanasia the muscles were reimaged ex vivo both quantitatively and qualitatively, either immediately, or after 1, 2, 3 or 5days of regeneration (each n=3 per group).Within 15 minutes of complement application, a rapid loss of CFP overlying the NMJ could be seen; in group A, the GFP signal remained unchanged, whereas in group B the GFP signal was also lost. In group C no changes to either CFP or GFP were observed. At 24h, 6% of the superficial NMJs in group A and 12% of the NMJs in group B exhibited CFP. In both groups, CFP returned within the next five days (group A: 93.5%, group B: 94%; p=0.739), with the recovery of CFP being preceded by a return of GFP-positive cells overlying the NMJ in group B. Auxiliary investigations revealed that the loss of CFP at the NMJ correlated with a loss of NT neurofilament immuno-reactivity and a return of CFP at the NMJ was accompanied by a return of neurofilament. In ultrastructural investigations, injured NTs were electron lucent and exhibited damaged mitochondria, a loss of filaments and a loss of synaptic vesicles. The examination of muscles after five days of regeneration revealed physiological NT-profiles.The results described above indicate that following a single anti-ganglioside Ab-mediated and complement-mediated attack, independent of whether there are healthy and mature perisynaptic Schwann cells overlying the NMJ, the murine NT is capable of recovering both its architectural and axolemmal integrity very rapidly. This data supports the notion that an equivalent mechanism may account for the rapid recovery seen in some clinical cases of AMAN.

Safety aspects of longitudinal administration of IGF-I/IGFBP-3 complex in neonatal mice

ObjectiveVery preterm birth is associated with a high risk of morbidity. Infants born very preterm have low serum levels of insulin-like growth factor I (IGF-I), that further decrease after birth. IGF-I is essential for brain development and low serum levels have been associated with retinopathy of prematurity. The present study aimed to investigate the effects of prolonged administration of a low dose of rhIGF-I/rhIGFBP-3 on glucose levels and total body weight, as well as liver, spleen and brain weights, and gray and subcortical white matter in newborn mice. DesignThe study was performed as three different trials. In all experiments C57BL/6N mice were injected with a rhIGF-I/rhIGFBP-3 complex or saline. In the first experimental trial, blood glucose levels were assessed 30min, 1h, 1.5h, 3h, 6h, 24h and 48h after the rhIGF-I/rhIGFBP-3 or saline injection on postnatal day (PND) 6. In the second trial, mice were injected daily from PND 3 to 11 and sacrificed on PND 12 for analysis of IGF-I serum levels. In the third trial, body and organ weights and effects on gray and white matter were assessed on PND 18 after PND 3–11 treatments as above. Effects on gray and white matter were measured using immunoreactivity for microtubule-associated protein-2 (MAP-2), myelin basic protein (MBP), 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNPase), neurofilament and oligodendrocyte lineage transcription factor 2 (Olig2). ResultsBlood glucose levels were unchanged in the rhIGF-I/rhIGFBP-3-treated group compared to baseline. In the control group glucose levels increased 30min after the second saline injection; levels were not elevated at the subsequent time point. Three hours after the rhIGF-I/rhIGFBP-3 or saline, glucose levels were lower in rhIGF-I/rhIGFBP-3-treated animals than in saline treated (p=0.026). At PND 18, total body weight was higher in rhIGF-I/rhIGFBP-3-treated mice compared with controls (p<0.05), but there were no differences between groups in brain, liver or spleen weights. No differences in gray matter area were found between groups. Analyses of white matter markers showed an increased number of Olig2-positive cells in rhIGF-I/rhIGFBP-3-treated mice compared with controls (p<0.001). There were no differences between groups in terms of MBP, CNPase or neurofilament immunoreactivity. ConclusionsProlonged administration of rhIGF-I/rhIGFBP-3 did not have a negative impact on blood glucose levels and was beneficial for total body growth.