Ubiquitin immunocytochemistry in human spongiform encephalopathies

The distribution of ubiquitin was studied by immunocytochemistry in eight cases of human spongiform encephalopathy and compared with the findings in seven age-and sex-matched cases of Alzheimer's disease and six non-demented control cases. The results were also compared with the immunocytochemical distribution of prion protein and the lysosomal aspartic protease cathepsin D. In the human spongiform encephalopathies, ubiquitin immunoreactivity was found in a punctate distribution at the periphery of prion protein amyloid plaques and in a finely granular pattern in the neuropil around and within areas of spongiform change. Cortical nerve cells contained scanty ubiquitinated dot-like inclusions, and occasional microglia around the areas of spongiform change also gave a positive staining reaction for ubiquitin, as did multiple irregular thread-like structures in the neuropil and white matter. The ubiquitin-containing structures at the plaque periphery in human spongiform encephalopathies resemble the neuritic processes at the periphery of the senile plaque in Alzheimer's disease. The granular positivity for ubiquitin associated with areas of spongiform change closely resembles the pattern of immunostaining seen with the antibodies to the prion protein and cathepsin D, consistent with the reported accumulation of ubiquitinated proteins and prion protein in lysosomes in the murine scrapie model. Further studies are required to investigate the role of lysosomes in this group of disorders, and to study the localization of other cell stress proteins and prion protein in spongiform encephalopathies.     

A morphometric and immunohistochemical study of the vestibular nuclear complex in bovine spongiform encephalopathy

Summary A morphometric and immunohistochemical study of the vestibular nuclear complex was performed on five bovine spongiform encephalopathy (BSE) and five control cow brains. Neurons of the lateral and superior vestibular nuclei were counted at 500-m intervals in 10-m-thick sections, using an image analysis system comprosing a projection microscope and digitising pad linked to a computer. A bimodal distribution of neuron diameters was recognised in the brains of normal cattle. One population of neurons had a mean diameter of 30 m and the other had a mean diameter of 60 m. The vestibular nuclei from BSE cattle had an approximately 50% reduction in total numbers of neurons when compared with controls (P<0.01). Cattle which were clinically diseased longer had the fewest number of neurons preserved. Diminshed numbers of neurons were detected throughout the area studied and affected neurons of all diameters. Immunohistochemical staining for synaptophysin a protein present in synapses throughout the CNS, showed no significant reduction in axon terminals synapsing with vestibular neurons, including vacuolated neurons of BSE brains, when controls and BSE brains were compared. This suggests that de-afferentation of neurons is not the cause of neuronal loss. Prion protein was detected in the neuropil of the vestibular nuclear complex of BSE brains but not control brains. These studies show that previously unsuspected neuronal loss is a significant feature of BSE.     

In vivo toxicity of prion protein in murine scrapie: ultrastructural and immunogold studies

Prion protein (PrP) is a cell surface, host coded, sialoglycoprotein which accumulates in excess in scrapie, Creutzfeldt‐Jakob disease, bovine spongiform encephalopathy and other transmissible spongiform encephalopathies. Infection of mice with the 87 V or ME7 scrapie strains results in distinctive and very different light microscopical patterns of vacuolation and disease specific PrP accumulation. In both of these scrapie strains immunogold electron microscopy was used to locate PrP to the plasmalemma of neurons from where it was released into the neuropil. Initial PrP accumulation around neurons and in early plaques lacking amyloid fibrils was generally not associated with morphological changes either of the neuron or dendrite releasing the PrP or in the adjacent neuropil in which excess PrP accumulated. However, accumulation of pre‐amyloid PrP in some brain areas was associated with specific degeneration of dendritic spines and axon terminals. Initial PrP aggregation into fibrils was also associated with tissue damage with both ME7 and 87 V plaques and diffuse accumulations. Tissue damage associated with fibrillogenesis was localized and would not be expected to have clinical significance. We conclude that pre‐amyloid PrP release and accumulation is not invariably toxic, either to the neuron releasing PrP or to the neuropil into which it is released. However, axon terminal degeneration and dendritic spine loss in some neuroanatomical areas may be indicative of specific PrP toxicity and may be the main cause of neurological dysfunction in murine scrapie.     

Cerebellar Purkinje cell loss in aging Hu-Bcl-2 transgenic mice

The number of cerebellar Purkinje cells is increased by over 40% in young transgenic mice that overexpress a human Bcl-2 transgene (Hu-Bcl-2). To determine whether the Bcl-2-mediated rescue of Purkinje cells persists through life, the numbers of Purkinje cells were estimated in 6-, 12-, 18-, and 24-month-old Hu-Bcl-2 transgenic mice and age-matched controls. In addition, the expression of four markers for Purkinje cell differentiation, calbindin (CaBP), the 67-kDa isoform of glutamic acid decarboxylase (GAD67), gamma-aminobutyric acid transaminase (GABA-T), and the NMDA-R1 receptor subtype (NMDA-NR1) was analyzed in 6-month-old Hu-Bcl-2 transgenics and controls to determine whether overexpression of Bcl-2 and rescue from naturally occurring cell death affects the normal differentiation of Purkinje cells. The estimates of Purkinje cell numbers showed that the number of Purkinje cells in the Hu-Bcl-2 transgenics declines after 6 months to approach wild-type values by 18 months. Although the exogenous human BCL-2 is still expressed in Purkinje cells at 24 months, the expression levels of human BCL-2 appear to decline significantly after 6 months, suggesting that survival of the supernumary Purkinje cells depends on the sustained overexpression of Bcl-2. All the Purkinje cells in the Hu-Bcl-2 transgenic mice appeared to express normal levels of the differentiation markers analyzed so there was no evidence for a class of Purkinje cells that do not differentiate normally when rescued from naturally occurring cell death.     

Prion protein genotype and pathological phenotype studies in sporadic Creutzfeldt-Jakob disease

A comparative semi-automated morphometric study was performed on the distribution of prion protein, spongiform change and astrocytosis in the brains of nine cases of sporadic Creutzfeldt-Jakob disease of differing genotype at the methionine-valine polymorphism at codon 129 of the prion protein gene. Custom-designed image analysis software was used to produce objective figures for each of the different pathological features throughout 13 different areas of the brain used for analysis. A significant positive correlation was observed between prion protein deposition and astrocytosis in all cases and no significant correlation was observed between spongiform change and prion protein deposition. Different patterns of pathology were found to relate to codon 129 genotype; valine homozygosity favoured the targeting of pathology to deep grey matter structures, while methionine homozygosity favoured cortical targeting of pathology. These results provide evidence that prion protein deposition is closely associated with an astrocytic reaction and suggest that codon 129 genotype may influence the pathological phenotype.     

Review: Membrane‐associated misfolded protein propagation in natural transmissible spongiform encephalopathies (TSEs), synthetic prion diseases and Alzheimer's disease

Protein misfolding has long been recognized as a primary cause of systemic amyloidosis and, increasingly, template‐mediated misfolding of native host proteins is now also considered to be central pathogenetic events in some neurodegenerative diseases. Alzheimer's disease, naturally occurring transmissible spongiform encephalopathies (TSEs) and experimental disorders caused by misfolded prion protein (PrP) generated in vitro all share an imbalance of protein synthesis, aggregation and clearance that leads to protein aggregation, prompting some to suggest that Alzheimer's disease is caused by a prion‐like mechanism. In TSEs, the host‐coded, glycosyl‐phosphoinositol (GPI) membrane‐anchored prion protein (PrP^c) is misfolded into disease‐associated, putatively infectious aggregates known as prions. In Alzheimer's disease the membrane‐spanning Alzheimer's precursor protein (APP) is progressively cleaved within the plasmalemma to form Aβ peptide fragments that can form pathogenic extracellular aggregates while microtubule‐associated tau proteins may also aggregate within neurones. Oligomeric Aβ peptides and full‐length misfolded PrP show a common potential to convert native protein and aggregate on plasma membranes before subsequent release to form amyloid fibrils in the extracellular space. However, the nature, membrane topography and processing of the precursor and propagated proteins in prion and Alzheimer's disease all differ, and each group of diseases has distinctive spectra of additional pathological changes and clinical signs suggesting that fundamentally different disease mechanisms are involved.     

Temporal-spatial pattern of acute neuronal and glial loss after spinal cord contusion

The secondary loss of neurons and glia over the first 24 h after spinal cord injury (SCI) contributes to the permanent functional deficits that are the unfortunate consequence of SCI. The progression of this acute secondary cell death in specific neuronal and glial populations has not previously been investigated in a quantitative manner. We used a well-characterized model of SCI to analyze the loss of ventral motoneurons (VMN) and ventral funicular astrocytes and oligodendrocytes at 15 min and 4, 8, and 24 h after an incomplete midthoracic contusion injury in the rat. We found that both the length of lesion and the length of spinal cord devoid of VMN increased in a time-dependent manner. The extent of VMN loss at specified distances rostral and caudal to the injury epicenter progressed symmetrically with time. Neuronal loss was accompanied by a loss of glial cells in ventral white matter that was significant at the epicenter by 4 h after injury. Oligodendrocyte loss followed the same temporal pattern as that of VMN while astrocyte loss was delayed. This information on the temporal-spatial pattern of cell loss can be used to investigate mechanisms involved in secondary injury of neurons and glia after SCI.     

Genetic influences on secondary degeneration and wound healing following spinal cord injury in various strains of mice

Various inbred strains of mice exhibit dramatic differences in sensitivity to excitotoxic cell death induced by systemic injections of kainic acid (KA). The present study evaluates whether the same strains are also differentially sensitive to secondary degeneration after spinal cord injury, in which excitotoxic cell death is thought to play a pathogenic role. Spinal cord crush injuries were produced at T9 in two inbred strains that are resistant to KA-induced excitotoxic cell death (C57Bl/6 and Balb/c) and four strains that are sensitive (CD-1, FVB/N, 129T2 Sv/EMS, and C57Bl/10). The spinal cord was prepared for light microscopy at intervals from 1 to 56 days postinjury, and the area of damaged tissue (termed lesion size) and amount of cavitation were determined by quantitative image analysis. Lesion size increased between 1 and 7 days in all strains and then decreased steadily in a wound-healing process that occurs uniquely in mice. The extent of cavitation also gradually decreased from 7 to 56 days in all strains. Although lesion area and cavitation decreased in all strains, there were significant differences in lesion size and cavitation across strains. Specifically, lesion areas in the KA-sensitive strains FVB/N, 129T2 Sv/EMS, and CD-1 were significantly larger at 56 days postinjury than in the KA-resistant strains C57Bl/6 and Balb/c. We conclude that the genetic differences that confer resistance and sensitivity to KA-induced neurotoxicity also modify the secondary degenerative processes that occur after spinal cord injury, so that resistance to excitotoxic injury leads to smaller overall lesions and a more effective wound-healing response.     

Ro5-4864 promotes neonatal motor neuron survival and nerve regeneration in adult rats

The translocator protein (18 kDa; TSPO), formerly known as the peripheral benzodiazepine receptor, is an outer mitochondrial membrane protein that associates with the mitochondrial permeability transition pore to regulate both steroidogenesis and apoptosis. TSPO expression is induced in adult dorsal root ganglion (DRG) sensory neurons after peripheral nerve injury and a TSPO receptor ligand, Ro5-4864, enhances DRG neurite growth in vitro and axonal regeneration in vivo . We have now found that TSPO is induced in neonatal motor neurons after peripheral nerve injury and have evaluated its involvement in neonatal and adult sensory and motor neuron survival, and in adult motor neuron regeneration. The TSPO ligand Ro5-4864 rescued cultured neonatal DRG neurons from nerve growth factor withdrawal-induced apoptosis and protected neonatal spinal cord motor neurons from death due to sciatic nerve axotomy. However, Ro5-4864 had only a small neuroprotective effect on adult facial motor neurons after axotomy, did not delay onset or prolong survival in SOD1 mutant mice, and failed to protect adult DRG neurons from sciatic nerve injury-induced death. In contrast, Ro5-4864 substantially enhanced adult facial motor neuron nerve regeneration and restoration of function after facial nerve axotomy. These data indicate a selective sensitivity of neonatal sensory and motor neurons to survival in response to Ro5-4864, which highlights that survival in injured immature neurons cannot necessarily predict success in adults. Furthermore, although Ro5-4864 is only a very weak promoter of survival in adult neurons, it significantly enhances regeneration and functional recovery in adults.     

Neurodegenerative changes and neuroapoptosis induced by systemic lipopolysaccharide administration are reversed by dexmedetomidine treatment in mice

Background: Sepsis-associated encephalopathy (SAE) is a frequent and nasty complication of sepsis, associated with patients increased risk of death and long-term brain dysfunctions.

Objective: This study aimed to explore the effect of dexmedetomidine (Dex), an anesthetic adjuvant, on the development of SAE.

Methods: Lipopolysaccharide (LPS, 10 mg/kg) was intraperitoneally injected to male BALB/c mice to induce sepsis. Dex (25 μg/kg) was given intraperitoneally immediately after LPS injection. Levels of TNF-α, IL-1β, malondialdehyde (MDA) and reactive oxygen species (ROS) were detected in mice brains tissue eight hours later after drug administration. Hematoxylin and eosin (HE) staining was used to detect brain pathologic change. We also detected apoptosis using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and Bcl-2, Bax, Caspase-3 expressions by western blot.

Results: Levels of TNF-α, IL-1β, MDA and ROS were increased in the brain tissue after LPS treatment, indicating that LPS injection resulted in increased brain inflammation and elevated oxidative stress. We further found a large quantity of degenerative neurons widespread in hippocampal CA1, CA3 regions and cerebral cortex according to HE staining. Dex could significantly decrease brain inflammation and oxidative stress by decreasing the levels of TNF-α, IL-1β, MDA and ROS, and ameliorate neurodegenerative changes. The associated results also demonstrated that Dex treatment ameliorated the LPS-induced neuronal apoptosis, probably by upregulating the Bcl-2 expression and downregulating the Bax expression.

Conclusion: Our results indicated that Dex could reverse neurodegenerative changes and neuroapoptosis in mice brain of septic mice induced by LPS through anti-inflammatory and antiapoptotic effects.     

Central neuronal loss and behavioral impairment in mice lacking neurotrophin receptor p75

The neurotrophin receptor p75 is a low-affinity receptor that binds neurotrophins. To investigate the role of p75 in the survival and function of central neurons, p75 null-mutant and wild type litter mate mice were tested on behavioral tasks. Null mutants showed significant performance deficits on water maze, inhibitory avoidance, motor activity, and habituation tasks that may be attributed to cognitive dysfunction or may represent a global sensorimotor impairment. The p75 null-mutant and wild type litter mate mice were assessed for central cholinergic deficit by using quantitative stereology to estimate the total neuronal number in basal forebrain and striatum and for subpopulations expressing the high-affinity tyrosine receptor kinase A (trkA) neurotrophin receptor and choline acetyltransferase (ChAT). In the adult brain, cholinergic neurons of the basal forebrain receive target-derived trophic support, whereas cholinergic striatal neurons do not. Adult p75 null-mutant mice had significant reduction of basal forebrain volume by 25% and had a corresponding significant loss of 37% of total basal forebrain neurons. The basal forebrain population of ChAT-positive neurons in p75-deficient mice declined significantly by 27%, whereas the trkA-positive population did not change significantly. There was no significant change in striatal volume or in striatal neuronal number either in total or by cholinergic subpopulation. These results demonstrate vulnerability to the lack of p75 in adult central neurons that are neurotrophin dependent. In addition, the loss of noncholinergic central neurons in mice lacking p75 suggests a role for p75 in cell survival by an as yet undetermined mechanism. Possible direct and indirect effects of p75 loss on neuronal survival are discussed. J. Comp. Neurol. 404:1–20, 1999. © 1999 Wiley-Liss, Inc.     

Cortical neuronal loss and hippocampal sclerosis are not detected by voxel‐based morphometry in individual epilepsy surgery patients

Voxel‐based morphometry (VBM) has detected differences between brains of groups of patients with epilepsy and controls, but the sensitivity for detecting subtle pathological changes in single subjects has not been established. The aim of the study was to test the sensitivity of VBM using statistical parametric mapping (SPM5) to detect hippocampal sclerosis (HS) and cortical neuronal loss in individual patients. T1‐weighted volumetric 1.5 T MR images from 13 patients with HS and laminar cortical neuronal loss were segmented, normalised and smoothed using SPM5. Both modulated and non‐modulated analyses were performed. Comparisons of one control subject against the rest (n = 23) were first performed to ascertain the smoothing level with the lowest number of SPM changes in controls. Each patient was then compared against the whole control group. The lowest number of SPM changes in control subjects was found at a smoothing level of 10 mm full width half maximum for modulated and non‐modulated data. In the patient group, no SPM abnormalities were found in the affected temporal lobe or hippocampus at this smoothing level. At lower smoothing levels there were numerous SPM findings in controls and patients. VBM did not detect any abnormalities associated with either laminar cortical neuronal loss or HS. This may be due to normalisation and smoothing of images and low statistical power in areas with larger inter‐individual differences. This suggests that the methodology may currently not be suitable to detect particular occult abnormalities possibly associated with seizure onset zone in individual epilepsy patients with unremarkable standard structural MRI. Hum Brain Mapp, 2009. © 2009 Wiley‐Liss, Inc.     

Mesenchymal stem cells facilitate axon sorting, myelination, and functional recovery in paralyzed mice deficient in Schwann cell-derived laminin

Peripheral nerve function depends on a regulated process of axon and Schwann cell development. Schwann cells interact with peripheral neurons to sort and ensheath individual axons. Ablation of laminin γ1 in the peripheral nervous system (PNS) arrests Schwann cell development prior to radial sorting of axons. Peripheral nerves of laminin-deficient animals are disorganized and hypomyelinated. In this study, sciatic nerves of laminin-deficient mice were treated with syngenic murine adipose-derived stem cells (ADSCs). ADSCs expressed laminin in vitro and in vivo following transplant into mutant sciatic nerves. ADSC-treatment of mutant nerves caused endogenous Schwann cells to differentiate past the point of developmental arrest to sort and myelinate axons. This was shown by (1) functional, (2) ultrastructural, and (3) immunohistochemical analysis. Treatment of laminin-deficient nerves with either soluble laminin or the immortalized laminin-expressing cell line 3T3/L1 did not overcome endogenous Schwann cell developmental arrest. In summary, these results indicate that (1) laminin-deficient Schwann cells can be rescued, (2) a cell-based approach is beneficial in comparison with soluble protein treatment, and (3) mesenchymal stem cells modify sciatic nerve function via trophic effects rather than transdifferentiation in this system.     

Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons

Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins,representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord.Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves.A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers.Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury.Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy.At 12 and 18 hours,and 1,2,3,4,5,and 6 days of injury,lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images.Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay.We found that in vivo electroporation did not affect caspase-3 expression in dorsal root ganglia.Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique.Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing.The results facilitate direct time course analysis of peripheral nerve axon regeneration.This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University,China(approval No.GLMC201503010)on March 7,2014.     

Axotomy-induced neuronal death and reactive astrogliosis in the lateral geniculate nucleus following a lesion of the visual cortex in the rat

Following a unilateral lesion of the visual cortex (cortical areas 17, 18, and 18a) in adult rats, neurons in the ipsilateral dorsal lateral geniculate nucleus (LGN) are axotomized, which leads to their atrophy and death. The time course of this neuronal degeneration was studied quantitatively, and the astroglial response was examined with glial fibrillary acidic protein immunohistochemistry. More than 95% of the neurons in the ipsilateral LGN survive during the first 3 days following a lesion of the visual cortex. However, in the next 4 days, massive neuronal death ensues, reducing the number of surviving neurons to approximately 33% of normal by the end of the first postoperative week. Between 2 weeks and 24 weeks postoperatively, the number of neurons present in the LGN declines very gradually from 34% to 17% of normal. Three days after a lesion of the visual cortex, the mean cross-sectional areas of ipsilateral LGN neurons are 13% smaller than normal (87%). By 1 week after the operation, surviving LGN neurons have atrophied to 66% of their normal area. Subsequently, the size of surviving neurons declines slowly to approximately 50% of normal at 24 weeks after the cortical lesion. Astrocytes in the ipsilateral LGN also react to cortical damage. At 1 day after a lesion of the visual cortex, glial fibrillary acidic protein immunoreactivity in the LGN is almost undetectable, but a distinct increase in immunoreactivity is seen at 3 days. Immunoreactivity peaks between 1 week and 2 weeks postoperatively and, thereafter, remains intense for at least 24 weeks. Thus, following a lesion of the visual cortex, the somata of neurons in the LGN remain essentially normal morphologically for about 3 days before the onset of rapid atrophy and death. Moreover, most of the neural cell death that occurs in the LGN after axotomy takes place in the last half of the first postoperative week. J. Comp. Neurol. 392:252–263, 1998. © 1998 Wiley-Liss, Inc.     

Apelin inhibits motor neuron apoptosis in the anterior horn following acute spinal cord and sciatic nerve injuries

Rat models of acute spinal cord injury and sciatic nerve injury were established.Apelin expression in spinal cord tissue was determined.In normal rat spinal cords,apelin expression was visible;however,2 hours post spinal cord injury,apelin expression peaked.Apelin expression increased 1 day post ligation of the sciatic nerve compared with normal rat spinal cords,and peaked at 3 days.Apelin expression was greater in the posterior horn compared with the anterior horn at each time point when compared with the normal group.The onset of neuronal apoptosis was significantly delayed following injection of apelin protein at the stump of the sciatic nerve,and the number of apoptotic cells after injury was reduced when compared with normal spinal cords.Our results indicate that apelin is expressed in the normal spinal cord and central nervous system after peripheral nerve injury.Apelin protein can reduce motor neuron apoptosis in the spinal cord anterior horn and delay the onset of apoptosis.