Regional differences in viral growth and central nervous system injury correlate with apoptosis

Infection of neonatal mice with reovirus T3 Dearing (T3D), the prototypic neurotropic reovirus, causes fatal encephalitis associated with neuronal injury and virus-induced apoptosis throughout the brain. T3D variant K (VarK) is an antigenic variant that has a nearly 1 million-fold reduction in neurovirulence following intracerebral (i.c.) inoculation compared to T3D and a restricted pattern of central nervous system injury with damage limited to the hippocampus, sparing other brain regions. We wished to determine whether the restricted pattern of VarK-induced injury was due to a reduced capacity to replicate in or injure cortical, as opposed to hippocampal, tissue. We found that following i.c. inoculation, VarK grew to similar titers as T3D in the hippocampus but had significantly lower titers in the cortex. Both viruses grew to identical titers and infected the same percentage of cells in mouse primary hippocampal cultures (MHC). In mouse primary cortical cultures (MCC) both the number of infected cells and the viral yield per infected cell were significantly lower for VarK than T3D. VarK-induced apoptosis was limited to the hippocampus in vivo, and in vitro both viruses induced apoptosis equally in MHC but VarK induced significantly less apoptosis than T3D in MCC. Growth of T3D in MCC was reduced to levels comparable to those of VarK following treatment of MCC with caspase inhibitors. Conversely, induction of apoptosis in VarK-infected MCC with fatty acid synthase-activating antibody significantly enhanced viral yield. These results suggest that the decreased neurovirulence of VarK may be due to its failure to efficiently induce apoptosis in cortical neurons.     

Deleterious effects of intestinal ischemia/reperfusion injury in the mouse enteric nervous system are associated with protein nitrosylation

Changes in intestinal function, notably impaired transit, following ischemia/reperfusion (I/R) injury are likely to derive, at least in part, from damage to the enteric nervous system. Currently, there is a lack of quantitative data and methods on which to base quantitation of changes that occur in enteric neurons. In the present work, we have investigated quantifiable changes in response to ischemia of the mouse small intestine followed by reperfusion from 1?h to 7?days. I/R caused distortion of nitric oxide synthase (NOS)-containing neurons, the appearance of a TUNEL reaction in neurons, protein nitrosylation and translocation of Hu protein. Protein nitrosylation was detected after 1?h and was detectable in 10% of neurons by 6?h in the ischemic region, indicating that reactive peroxynitrites are rapidly produced and can interact with proteins soon after reperfusion. Apoptosis, revealed by TUNEL staining, was apparent at 6?h. The profile sizes of NOS neurons were increased by 60% at 2?days and neurons were still swollen at 7?days, both in the ischemic region and proximal to the ischemia. The distribution of the enteric neuron marker and oligonucleotide binding protein, Hu, was significantly changed in both regions. Hu protein translocation to the nucleus was apparent by 3?h and persisted for up to 7?days. Particulate Hu immunoreactivity was observed in the ganglia 3?h after I/R but was never observed in control. Our observations indicate that effects of I/R injury can be detected after 1?h and that neuronal changes persist to at least 7?days. Involvement of NO and reactive oxygen species in the changes is indicated by the accumulation of nitrosylated protein aggregates and the swelling and distortion of nitrergic neurons. It is concluded that damage to the enteric nervous system, which is likely to contribute to functional deficits following ischemia and re-oxygenation in the intestine, can be quantified by Hu protein translocation, protein nitrosylation, swelling of nitrergic neurons and apoptosis.     

Glycoproteins associated with myelin in the central nervous system

Purified myelin fractions from the central nervous system contain one major myelin-associated glycoprotein and approximately 16 minor glycoproteins. While the genuine association of the major myelin-associated glycoprotein with the oligodendroglial myelin unit is demonstrated, the possibility exists that several of the minor glycoproteins have their origin in contaminating membranes not related to myelin. The major myelin-associated glycoprotein is probably not present in compacted myelin, but immunocytochemical and subfractionation studies indicate that it is confined to the periaxonal and paranodal region of the myelin sheath. In experimental demyelination and multiple sclerosis, the major glycoprotein is the first myelin constituent to be affected. Its localization on the membrane surface where myelin and axolemma are in close contact, and other indirect evidence indicate that the major glycoprotein, and possibly other myelin-associated glycoproteins, could play a role in the process of myelination and myelin maintenance.     

Motor skill training and strength training are associated with different plastic changes in the central nervous system.

Changes in corticospinal excitability induced by 4 wk of heavy strength training or visuomotor skill learning were investigated in 24 healthy human subjects. Measurements of the input-output relation for biceps brachii motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation were obtained at rest and during voluntary contraction in the course of the training. The training paradigms induced specific changes in the motor performance capacity of the subjects. The strength training group increased maximal dynamic and isometric muscle strength by 31% (P < 0.001) and 12.5% (P = 0.045), respectively. The skill learning group improved skill performance significantly (P < 0.001). With one training bout, the only significant change in transcranial magnetic stimulation parameters was an increase in skill learning group maximal MEP level (MEP(max)) at rest (P = 0.02) for subjects performing skill training. With repeated skill training three times per week for 4 wk, MEP(max) increased and the minimal stimulation intensity required to elicit MEPs decreased significantly at rest and during contraction (P < 0.05). In contrast, MEP(max) and the slope of the input-output relation both decreased significantly at rest but not during contraction in the strength-trained subjects (P < or = 0.01). No significant changes were observed in a control group. A significant correlation between changes in neurophysiological parameters and motor performance was observed for skill learning but not strength training. The data show that increased corticospinal excitability may develop over several weeks of skill training and indicate that these changes may be of importance for task acquisition. Because strength training was not accompanied by similar changes, the data suggest that different adaptive changes are involved in neural adaptation to strength training.     

Inflammation in central nervous system injury

Inflammation is a key component of host defence responses to peripheral inflammation and injury, but it is now also recognized as a major contributor to diverse, acute and chronic central nervous system (CNS) disorders. Expression of inflammatory mediators including complement, adhesion molecules, cyclooxygenase enzymes and their products and cytokines is increased in experimental and clinical neurodegenerative disease, and intervention studies in experimental animals suggest that several of these factors contribute directly to neuronal injury. Most notably, specific cytokines, such as interleukin-1 (IL-1), have been implicated heavily in acute neurodegeneration, such as stroke and head injury. In spite of their diverse presentation, common inflammatory mechanisms may contribute to many neurodegenerative disorders and in some (e.g. multiple sclerosis) inflammatory modulators are in clinical use. Inflammation may have beneficial as well as detrimental actions in the CNS, particularly in repair and recovery. Nevertheless, several anti-inflammatory targets have been identified as putative treatments for CNS disorders, initially in acute conditions, but which may also be appropriate to chronic neurodegenerative conditions.     

Pnn and SR family proteins are differentially expressed in mouse central nervous system

Pinin (pnn) is an SR-related protein that is ubiquitously expressed in most cell types and functions in regulating pre-mRNA splicing and mRNA export. Previously, we demonstrated that pnn is expressed in all tissues during mouse embryonic development with highest levels of expression in the central nervous system (CNS). Here we show that pnn and other SR proteins including SC35 are differentially expressed in the adult mouse CNS, displaying cell type-specific distribution patterns. Immunohistochemical analysis of whole-brain sections showed that levels of pnn and SR proteins expression were very low or nonexistent in the corpus callosum and white matter of cerebellum and spinal cord. Double-immunostaining with antibodies specific to neuron or glial cells showed that most astrocytes and microglia expressed neither pnn nor SR proteins. In contrast, oligodendrocytes and neurons expressed moderate and high levels, respectively, of both pnn and SR proteins. These results suggest that astrocytes are unique among cell types of neuroblast origin in terms of expression SR family proteins. Our results pave the way for future studies of the functional roles of pnn and SR family proteins in adults.     

Transgenic mouse model for central nervous system demyelination.

A common feature of demyelinating diseases such as multiple sclerosis in humans and experimental autoimmune encephalomyelitis in rodents is the marked elevation in the expression of the major histocompatibility complex (MHC) antigens in the involved sites. By specific targeting of a syngeneic MHC class I gene to oligodendrocytes, we have generated transgenic mice which not only exhibit severe involuntary tremors and develop tonic seizures but also show extensive demyelination in both the brain and the spinal cord. The fact that demyelination in these mice occurs in the absence of immune infiltration dismisses an autoimmune involvement but suggests that the MHC class I antigens play a direct role in inducing disease. Our findings lend support to the possibility that demyelinating diseases are induced by infectious agents such as viruses which can either directly activate MHC gene expression in oligodendroglia or indirectly activate expression through the release by reactive T cells of gamma interferon in the brain.     

Mycoplasma hominis infection of the central nervous system in a neonate.

A newborn infant with group B streptococcal sepsis responded to the intravenous administration of antibiotics within 3 days, but then began to show signs of central nervous system infection. Routine cultures of cerebrospinal fluid samples were negative, but others done for Mycoplasma hominis yielded that organism. After 3 more days of antibiotic therapy, cultures for M. hominis were negative, and the child''s recovery was uneventful.     

Neuroplasticity. Key to recovery after central nervous system injury.

After an injury to the central nervous system, physical and cognitive impairments and disabilities often abate. These gains may be partly mediated by mechanisms that allow reorganizing of the structure and function within gray and white matter. The potential to enhance neurologic recovery by manipulating the brain and spinal cord must now be considered in clinical practice. Today''s rehabilitation routines may not encourage maximum recovery. Indeed, some commonly used physical and pharmacologic methods could inhibit the restoration of motor activities such as walking. On the other hand, therapies that use our expanding knowledge of neuroplasticity could lead to better results for patients.     

Behavioral recovery associated with central nervous system regeneration in the snail Melampus

The pulmonate snail Melampus bidentatus regenerates central nervous tracts following commissurotomy, connective transection, and cerebral ganglion ablation. Our goal was to determine whether or not neural regrowth within the central nervous system restored behaviors disrupted by lesions. One behavior that is disrupted by commissurotomy is retraction of facial structures that are contralateral to a stimulated facial region, a response that normally accompanies the ipsilateral retraction. Tentacle withdrawal on the side contralateral to stimulation reappeared on a timescale that was correlated with growth of a commissural link (8-19 days post-lesion). Electrophysiological recordings from a labial nerve pathway that has a contralateral component similar to the contralateral tentacle response showed that development or strengthening of an alternative pathway could also mediate contralateral responses. Thus, a major conclusion of this study was that both tract regeneration and changes in existing CNS pathways can underlie recovery. The percentage (approx. 75%) of snails that regenerate the cerebral commissure and show behavioral recovery is established early in the period following commissure transection. Behavioral recovery and anatomical evidence of regeneration were also correlated in the other two operations: single cerebral ganglion removal and unilateral cerebropleural and cerebropedal connective transection. We conclude that Melampus is able to regenerate neuronal connectivity that can restore normal behavior.     

In vivo electroporation in the embryonic mouse central nervous system

This protocol describes a basic method for in vivo electroporation in the nervous system of embryonic mice. Delivery of electric pulses following microinjection of DNA into the brain ventricle or the spinal cord central canal enables efficient transfection of genes into the nervous system. Transfection is facilitated by forceps-type electrodes, which hold the uterus and/or the yolk sac containing the embryo. More than ten embryos in a single pregnant mouse can be operated on within 30 min. More than 90% of operated embryos survive and more than 90% of these survivors express the transfected genes appropriately. Gene expression in neurons persists for a long time, even at postnatal stages, after electroporation. Thus, this method could be used to analyze roles of genes not only in embryonic development but also in higher order function of the nervous system, such as learning.     

Peripheral Taenia infection increases immunoglobulins in the central nervous system

Human cysticercosis is a disease caused by larvae of the cestode Taenia solium. It is an important common cause of adult-onset seizures world-wide where it exacts a debilitating toll on the health and well-being of affected communities. It is commonly assumed that the major symptoms associated with cysticercosis are a result of the direct presence of larvae in the brain. As a result, the possible effects of peripherally located larvae on the central nervous system are not well understood. To address this question, we utilised the Taenia crassiceps intra-peritoneal murine model of cysticercosis, where larvae are restricted to the peritoneal cavity. In this model, previous research has observed behavioural changes in rodents but not the development of seizures. Here we used ELISAs, immunoblotting and the Evans Blue test for blood–brain barrier permeability to explore the central effects of peripheral infection of mice with T. crassiceps. We identified high levels of parasite-targeting immunoglobulins in the sera of T. crassiceps-infected mice. We show that the T. crassciceps larvae themselves also contain and release host immunoglobulins over time. Additionally, we describe, for the first known time, significantly increased levels of IgG within the hippocampi of infected mice, which are accompanied by changes in blood–brain barrier permeability. However, these T. crassiceps-induced changes were not accompanied by alterations to the levels of proinflammatory, pro-seizure cytokines in the hippocampus. These findings contribute to the understanding of systemic and neuroimmune responses in the T. crassiceps model of cysticercosis, with implications for the pathogenesis of human cysticercosis.     

Expression of Frizzled10 in mouse central nervous system

Frizzled transmembrane proteins (Fzd) are receptors of Wnts, and they play key roles during central nervous system (CNS) development in vertebrates. Here we report the expression pattern of Frizzled10 in mouse CNS from embryonic stages to adulthood. Frizzled10 is expressed strongly at embryonic days E8.5 and E9.5 in the neural tube and tail bud. At E10.5, Frizzled10 is expressed in the forebrain vesicle, the fourth ventricle and the dorsal spinal cord. From E12.5 to E16.5, Frizzled10 expression is mainly observed in the cortical hem/fimbria, the neuroepithelium of the third ventricular zone, midbrain, developing cerebellum, and dorsal spinal cord. At P0, with the exception of expression in the fimbria, Frizzled10 mRNA expression is limited to specific nuclei including the ventral posterior thalamic nucleus (VP) and the dorsal lateral geniculate nucleus (DLG) in the developing thalamus as well as in the proliferative ventricular zone of the developing cerebellum. From P20 to adult, Frizzled10 mRNA is detected only in the internal capsule (ic). Our data show that expression of Frizzled10 is very strong during embryonic development of the CNS and suggest that Frizzled10 may play an essential role in spatial and temporal regulation during neural development.     

A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system meylin.

Purified myelin fractions prepared from young adult rat brain contain a novel sphingomyelinase which has a pH optimum of 7.0 and does not require divalent cations. This sphingomyelinase is different from the two previously known sphingomyelinases in the brain--the acidic sphingomyelinase and the magnesium-dependent neutral sphingomyelinase. When the distributions of the sphingomyelinases among the purified myelin, the total subcellular fractions heavier than myelin (greater than 0.85 M sucrose), and the microsomes were examined, the magnesium-independent sphingomyelinase was detected only in myelin, while the magnesium-dependent sphingomyelinase was present in the other two fractions but not in myelin. Therefore, this new sphingomyelinase appears to be specifically localized in the myelin sheath.     

Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system

Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.