Regulation of nestin expression after cortical ablation in adult rat brain.

During embryogenesis, transient expression of nestin in proliferating neuroepithelial stem cells signals the commitment of progenitor cells to differentiate. Although adult mammalian brain contains very little nestin, significant upregulation of nestin has been reported following cerebral injury, leading to speculation that nestin may be involved in brain repair. In this study, we assessed the temporal profile of nestin expression following ablation injury of the sensory barrel cortex and investigated the influence of contralateral whisker stimulation on nestin expression. Since the adult mammalian brain contains proliferating neuronal progenitor cells that can be labeled with bromodeoxyuridine (BrdU), we also determined the association of nestin reexpression with BrdU-labeled cells. Nestin reexpression was detected predominantly in the ipsilateral cortex 3 days post-ablation. There was no significant nestin upregulation in the subcortical region. Nestin reexpression was most marked surrounding the lesion, but also extended throughout the entire lateral cortex. Nestin in the ipsilateral cortex subsided by day 7, although perilesional nestin expression was still apparent 28 days post-injury. Western blot analysis of nestin expression 3 days post-ablation confirmed a significant two-fold increase in nestin expression (p<0.05). Double immunofluorescence labeling demonstrated that the majority of nestin expression occurred in astrocytes. We were unable to detect any colocalization with neuronal makers. However, BrdU-labeled cells, which were readily detected in the subventricular zone prior to injury, were readily detected in the perilesional area 3 days post-ablation, concomitant with nestin in this area. Confocal microscopy detected several BrdU-positive cells expressing nestin. Taken together, the data support a potential role for nestin reexpression in brain repair.

Cortical spreading depression transiently activates MAP kinases.

Cortical spreading depression (CSD) has been shown to have neuroprotective effects when administered in advance of cerebral ischemia. The mechanism by which CSD induces its neuroprotective effect however remains to be elucidated. Since MAP kinases have been shown to impart neuroprotection in ischemic preconditioning paradigms, we attempted to determine the role CSD may have in the activation of MAPK. We show that CSD is capable of increasing the phosphorylation of ERK in a MEK-dependent manner. This phosphorylation is, however, transient, as phosphorylated ERK levels return to control levels 45 min after 2 h of CSD elicitation. Immunohistochemical analysis reveals that the phosphorylated form of ERK is located ubiquitously in cells of the CSD-treated cortex while CSD-elicited MEK phosphorylation resides solely in the nuclei. These data suggest that CSD may act via the MAP kinase pathways to mediate preconditioning.

Attenuation of neurotoxicity in cortical cultures and hippocampal slices from E2F1 knockout mice.

The E2F1 transcription factor modulates neuronal apoptosis induced by staurosporine, DNA damage and beta-amyloid. We demonstrate E2F1 involvement in neuronal death induced by the more physiological oxygen-glucose deprivation (OGD) in mouse cortical cultures and by anoxia in mouse hippocampal slices. E2F1(+/+) and (-/-) cultures were comparable, in that they contained similar neuronal densities, responded with similar increases in intracellular calcium concentration ([Ca(2+)]i) to glutamate receptor agonists, and showed similar NMDA receptor subunit mRNA expression levels for NR1, NR2A and NR2B. Despite these similarities, E2F1(-/-) cultures were significantly less susceptible to neuronal death than E2F1(+/+) cultures 24 and 48 h following 120-180 min of OGD. Furthermore, the absence of E2F1 significantly improved the ability of CA1 neurons in hippocampal slices to recover synaptic transmission following a transient anoxic insult in vitro. These results, along with our finding that E2F1 mRNA levels are significantly increased following OGD, support a role for E2F1 in the modulation of OGD- and anoxia-induced neuronal death. These findings are consistent with studies showing that overexpression of E2F1 in postmitotic neurons causes neuronal degeneration and the absence of E2F1 decreases infarct volume following cerebral ischemia.

Cyclin-dependent kinases as a therapeutic target for stroke.

Cyclin-dependent kinases (CDKs) are commonly known to regulate cell proliferation. However, previous reports suggest that in cultured postmitotic neurons, activation of CDKs is a signal for death rather than cell division. We determined whether CDK activation occurs in mature adult neurons during focal stroke in vivo and whether this signal was required for neuronal death after reperfusion injury. Cdk4/cyclin D1 levels and phosphorylation of its substrate retinoblastoma protein (pRb) increase after stroke. Deregulated levels of E2F1, a transcription factor regulated by pRb, are also observed. Administration of a CDK inhibitor blocks pRb phosphorylation and the increase in E2F1 levels and dramatically reduces neuronal death by 80%. These results indicate that CDKs are an important therapeutic target for the treatment of reperfusion injury after ischemia.

Cervical artery dissection is associated with widened aortic root diameter.

OBJECTIVE: Dissection of the internal carotid and vertebral arteries is a well recognized cause of stroke, especially in the middle-aged. The exact etiology of this condition is controversial. According to one theory there is an underlying vasculopathy originating from disturbed development of the neural crest. The neural crest gives rise to several tissues, including the tunica media of large cervical arteries and the outflow tract of the heart. We attempted to test the theory that developmental abnormality at the level of the neural crest may play a role in dissection of the large cervical arteries. METHODS: We designed a retrospective case control study. By means of transthoracic echocardiography we measured the aortic root diameter in a group of patients with radiographically determined dissection of at least one large artery in the neck. The results were compared to a control group. RESULTS: In comparison to age matched controls, male patients were found to have a significantly larger aortic root. Although a similar trend was apparent in females, the difference between the patient and control group of females was not statistically significant. CONCLUSIONS: Patients with cervical artery dissections may have other abnormalities in other organs arising from the neural crest. A larger prospective clinical study and further research are needed to establish a firm link between dissection of the cervical arteries and abnormalities in other organs.

Cortical spreading depression activates trophic factor expression in neurons and astrocytes and protects against subsequent focal brain ischemia.

We recently reported that cortical spreading depression (CSD), used to precondition rat brain, reduced cortical infarction volume resulting from focal cerebral ischemia by middle cerebral artery occlusion (MCAO) 3 days later. The mechanisms underlying this protective effect by CSD remains to be explored. In this study, we confirm that CSD is neuroprotective when KCl is applied epidurally rather than intracortically. Neocortical infarct volume was 101.3+/-48.5 mm3 and 45.3+/-44.1 mm3 in the sham and CSD group, respectively (p<0.05). Using image analysis, we identified the cortical region spared from infarction by the prior CSD. We then determined the distribution of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) mRNA and the time course of their expression in groups of animals treated with CSD and their controls. We also examined the response of astrocytes to CSD using glial fibrillary acidic protein (GFAP) as a marker. In situ hybridization (done at 0, 3, 12, 24, 72 or 168 h after CSD) showed significant elevation of BDNF mRNA in the cortex immediately after CSD in a distribution surrounding the spared cortex, while bFGF mRNA rose 12 h after CSD and appeared more within the core of the ischemic region. Immunohistochemistry (done at 1, 3 or 7 days after CSD) demonstrated GFAP in the neocortex, with a peak at 3 days after CSD. Heat shock protein 72 (HSP72) expression was not affected by CSD. We concluded that upregulation of trophic factors and activation of glial cells may contribute to the neuroprotection induced by CSD.

Ischemic penumbra: the therapeutic window.

The concept of ischemic penumbra has extended our understanding of the focal ischemic process and in many ways has revitalized our research. Simply stated, the penumbra is the part of the brain that is sandwiched between brain regions committed to die and those that receive enough blood to communicate. Therefore, it is ischemic brain tissue that has just enough energy to survive for a short time but not enough to communicate and function. The life expectancy of the penumbra is short. Although the penumbra is an elegant concept, in practice it has been a difficult one to exploit. For one thing, it is unstable in both time and space. Depending on the severity and the duration of the focal ischemia, it may be anywhere in the ischemic brain. We believe that nimodipine binding experiments have taught us a great deal about the ischemic penumbra. Second, cells in the penumbra may die not by necrosis but by apoptosis. If that is true, then our concepts about the benign transient ischemic attack may need revision. Third, the penumbra may regain its ability to survive not only through reperfusion but also by interrupting the process of commitment to apoptosis.

Sectional impressions for mandibular distal extension removable partial dentures.

STATEMENT OF PROBLEM: Corrected impression procedures provide the best possible support to be gained from an edentulous ridge for a removable partial denture, but they are time-consuming and technique-sensitive. PURPOSE: An experimental mandibular distal extension removable partial denture was used to measure the vertical movement of denture bases during biting. MATERIAL AND METHODS: Five patients participated for whom vertical movement of denture bases was measured by comparing the thickness of plaster interocclusal records made with the denture at rest and under biting pressure. Denture bases were processed on master casts formed from final impressions made in full arch custom trays. Secondly, bases were readapted using sectional relining impressions with the same regular body polysulfide impression material. RESULTS: Statistical analysis of the vertical movement of denture bases during biting at 4 points on the right and left denture bases showed a significant decrease in vertical movement with sectional impression. The reduction was less than a 0.1 mm, a result that questions the routine use of sectional impression for every distal-extension mandibular removable partial denture. CONCLUSION: Supportive form of distal-extension edentulous ridges can be recorded successfully by full arch impressions in custom trays.

Regional activation of L-type voltage-sensitive calcium channels in experimental thiamine deficiency.

During pyrithiamine-induced thiamine deficiency (PTD), specific regions of the brain develop histological damage. The basis of this selective vulnerability is unknown but the mechanism may involve a glutamate-mediated excitotoxic process in affected structures, leading to alterations in membrane potential and disturbances in calcium homeostasis. In this study, we have examined the volume of distribution of [3H]nimodipine, an L-type voltage-sensitive calcium channel (VSCC) antagonist, in the brain of the PTD rat. An increase in specific binding of [3H]nimodipine was detected only in the posterior thalamus at the symptomatic stage, immediately following the loss of righting reflexes (P < 0.0001). There was also an increase in nonspecific binding in the medial geniculate and inferior colliculi. Replenishment with thiamine at the symptomatic stage returned [3H]nimodipine binding to normal levels. These findings provide evidence that depolarization and activation of L-type VSCCs occur in the posterior thalamus and may contribute to the appearance of histological lesions in this structure during experimental thiamine deficiency.

Hippocampal Myc and p53 expression following transient global ischemia.

The proto-oncogene c-myc, and the tumor suppressor gene p53, encode proteins which function as transcriptional regulating factors governing cell proliferation, differentiation, and apoptosis. Recent evidence suggests that the delayed neuronal death which follows an episode of transient forebrain ischemia may involve apoptotic processes. We have therefore utilized immunohistochemistry to investigate the effects of transient global ischemia on neuronal expression of p53- and Myc-like immunoreactivities in the rodent forebrain 2, 12, 24, 48, and 72 h following reperfusion. Transient global ischemia (20 min), produced by four vessel occlusion (4-VO), initially elevated p53-like immunoreactivity in both CA1 and CA3 hippocampal subfields at 24 h of recirculation. However, distinct patterns of gene expression became evident in these regions at later time points. A pivotal difference was the persistence of ischemia-induced increases of p53- and Myc-like immunoreactivity in the CA1 region of the hippocampus. Unlike CA3 neurons where p53-like immunoreactivity subsided to basal levels by 48 h of survival, CA1 neurons continued to display increased p53-immunoreactivity 48 h post-ischemia, while Myc-like immunoreactivity was selectively elevated in CA1 neurons at this time point. Ischemia-induced increases in p53-like immunoreactivity were also detected in vulnerable regions of the amygdala, thalamus, and cortex 12 to 48 h after recirculation. Given that both p53 and Myc have been implicated in gene signalling pathways which mediate programmed cell death, our findings which demonstrate that 4-VO produces persistent elevations of p53- and Myc-like immunoreactivities in vulnerable neurons suggest that these proteins may also contribute to delayed neuronal death following an episode of transient forebrain ischemia.

Ischemia-induced CA1 neuronal death is preceded by elevated FosB and Jun expression and reduced NGFI-A and JunB levels.

Alterations in levels of the immediate-early gene (IEG) proteins Fos, FosB, DeltaFosB, Jun, JunB, JunD, and NGFI-A were investigated in rat hippocampus by immunohistochemistry 2, 12, 24, and 48 h after forebrain ischemia. Transient global ischemia of 20 min, produced by four vessel occlusion (4-VO), elicited different patterns of IEG expression in vulnerable CA1 and more resilient CA3 neurons. Cell counts revealed that except for JunD and NGFI-A, immunoreactivity for all examined IEGs was initially elevated by forebrain ischemia in both CA1 and CA3 hippocampal subfields. However, distinct patterns of IEG expression became evident in these regions at later time points. The pivotal difference was the persistence of ischemia-induced elevations of FosB and Jun expression in the CA1 region of the hippocampus. Unlike CA3 neurons, where IEG immunoreactivity had subsided to basal levels by 24-48 h, CA1 neurons continued to display increased FosB- and Jun-like immunoreactivity 48 h post-ischemia. Western blot analysis revealed that elevated expression of both FosB and DeltaFosB-like proteins were responsible for the immunohistochemical detection of enhanced FosB-like immunoreactivity in CA1 neurons at 48 h. These findings are consistent with recent in vitro studies that implicate FosB and Jun in gene signalling pathways responsible for programmed cell death. In contrast to FosB and Jun, JunB expression declined significantly below basal levels in CA1 neurons at 48 h, yet remained unaltered in CA3 neurons. Given that JunB can inhibit the transactivating properties of Jun, decreased JunB levels may contribute to the apoptotic death of CA1 neurons by enhancing the transcriptional regulating activity of Jun. Also notable at 48 h was the complete loss of constitutive NGFI-A expression from CA1 neurons of ischemic animals. These findings suggest that persistent elevations in FosB and Jun expression, concurrent with reductions in JunB and NGFI-A levels, contribute to the apoptotic death of CA1 neurons after forebrain ischemia.

Immediate-early gene expression in the brain of the thiamine-deficient rat.

Pyrithiamine-induced thiamine deficiency (PTD) in the rat is associated with neuronal loss in the thalamus and inferior colliculus. Recently, we were able to demonstrate the occurrence of apoptosis in the thalamus of these animals. Given that immediate-early genes (IEGs) participate in signal transduction pathways that mediate programmed cell death, the present study utilized in situ hybridization and immunohistochemistry to examine the expression of four IEGs (c-fos, c-jun, fos-B, and NGFI-A) during the progression of PTD. Elevated c-fos mRNA levels were initially observed in the posterior medial thalamus on d 12 of the deficiency. At the acute symptomatic stage (characterized by a loss of righting reflex on d 16-17), the posterior-medial thalamus exhibited increased mRNA for all genes examined, whereas the inferior colliculus demonstrated mRNA induction for c-fos, c-jun, and NGFI-A. Immunohistochemical analysis revealed that elevations of IEG mRNA associated with the acute symptomatic stage were consistently translated into protein in the thalamus. In contrast, whereas elevated Fos- and Jun-like immunoreactivity were detected in the inferior colliculus at this stage, NGFI-A-like immunoreactivity declined significantly below basal levels, suggesting a translational block. These results are consistent with our recent findings of apoptotic cell death, and indicate that differential patterns of IEG expression occur in the thalamus and inferior colliculus during PTD, which may contribute to the pathogenesis of this disorder.

Nestin expression in reactive astrocytes following focal cerebral ischemia in rats.

During central nervous system (CNS) development, intermediate filaments are subjected to a sequential remodelling process. Nestin is a distinct intermediate filament which is transiently expressed in proliferating neuroepithelial stem cells during the neurulation stage of development. Nestin re-expression in the adult rat was studied following transient (2 h) middle cerebral artery occlusion. Seven days after the ischemic insult, nestin reactive astrocytes were found in the border zone surrounding cerebral infarction. Nestin immunoreactivity delineated a zone between infarction and the surrounding intact cerebral parenchyma. In situ hybridization for nestin mRNA showed early changes in small cells in the surround of the ischemic lesion. These results with nestin, along with other stem cell markers expressed by reactive astrocytes, suggest an embryonic reversion of the mature cytoskeleton as a response of astrocytes to cerebral injury.

Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus.

We show here that transient forebrain ischemia selectively elevates levels of neuronal apoptosis inhibitory protein (NAIP) in rat neurons that are resistant to the injurious effects of this treatment. This observation suggests that increasing NAIP levels may confer protection against ischemic cell death. Consistent with this proposal, we demonstrate that two other treatments that increase neuronal NAIP levels, systemic administration of the bacterial alkaloid K252a and intracerebral injection of an adenovirus vector capable of overexpressing NAIP in vivo, reduce ischemic damage in the rat hippocampus. Taken together, these findings suggest that NAIP may play a key role in conferring resistance to ischemic damage and that treatments that elevate neuronal levels of this antiapoptotic protein may have utility in the treatment of stroke.

In vivo uptake of [3H]nimodipine into brain during cortical spreading depression.

We report autoradiographic measurements of the in vivo uptake of [3H]nimodipine during the nonischemic depolarization of cortical spreading depression (CSD) in rat brain. [3H]Nimodipine uptake in brain was determined regionally in rats undergoing CSD (n = 8) and was significantly increased in cortex (14 +/- 7%) and hippocampus (10 +/- 6%) on the stimulated side relative to the contralateral hemisphere when compared with the same measurements in a control group (n = 8). A similar measurement using the physiologically inert radiotracer [14C]iodoantipyrine to control for potential effects of CSD on radioligand distribution showed a minimal increase (2.4 +/- 0.7%) of radiotracer uptake in cortex after CSD. This increase was significantly less than that observed in the [3H]nimodipine uptake studies. We hypothesize that increased in vivo [3H]nimodipine uptake in CSD identifies regions of depolarization and thus infers activation of the L-type voltage sensitive calcium channels.

Apoptosis is restricted to the thalamus in thiamine-deficient rats.

Thiamine deficiency (TD) produces lesions in the thalamus, mamillary and medial geniculate nuclei, and inferior colliculus. To clarify the pathogenesis of these lesions, we examined the occurrence of hallmarks of apoptosis following TD in rat brain. Histological assessment showed apoptotic cells in the thalamus and medial geniculate nucleus but not in the inferior colliculus. We used terminal deoxynucleotidyl transferase-mediated deoxyuridine (dUPT)-biotin nick-end labelling (TUNEL) and gel electrophoresis to demonstrate that TD is associated with apoptotic cell death. In the thalamus, DNA fragmentation appeared from day 14 of deficiency and preceded the appearance of ataxia. The inferior colliculus and mamillary nucleus were without electrophoretic DNA fragments, and only rare TUNEL-positive labelling was observed. This model shows a rare combination of both apoptosis and necrosis in the same lesioned brain.

Subtle neuronal death in striatum after short forebrain ischemia in rats detected by in situ end-labeling for DNA damage.

BACKGROUND AND PURPOSE: Neuronal cell death after global brain ischemia occurs predominantly by necrosis, whereas only a minor fraction of cell death may occur through apoptosis. Brief or moderate insults are thought to facilitate apoptosis, which is associated with DNA fragmentation. After 10 minutes of four-vessel occlusion in rats, conventional neuropathological analysis shows neuronal cell death in hippocampal CA1 but not in the striatum. Thus, we compared hippocampus and striatum for occurrence of cells with DNA fragmentation. METHODS: A brief insult of 10 minutes of forebrain ischemia was induced in rats using four-vessel occlusion, and groups of brains were studied at 1, 3, 6, and 12 hours and at 1, 3, and 7 days after ischemia. In situ end-labeling (ISEL) was used to detect neurons undergoing DNA fragmentation. The hippocampal CA1 area was compared with the striatum. Conventional staining and immunohistochemical markers served to exclude ischemic neuronal cell death in the striatum. RESULTS: Hippocampal CA1 neurons were ISEL-positive by 3 days after ischemia. In contrast, positive cells became evident in the striatum between 3 hours to 3 days after ischemia. The ISEL-positive cells were scattered throughout the striatum with a preference for the dorsomedial areas and accounted for about 0.2% of the neurons per striatal area at 1 day. Conventional staining and immunohistochemical markers failed to reveal areas of overt cell damage in the striatum. CONCLUSIONS: The scattered cell damage in the striatum after brief forebrain ischemia suggests the occurrence of an apoptotic process. The striatum therefore may be prone to subtle cell death due to metabolic insults.

Stomatognathic dysfunctional symptoms in Saudi Arabian adolescents.

Secondary school Saudi students participated in a questionnaire about stomatognathic dysfunction symptoms. The adolescents were interviewed about general health, peripheral joint disease, chewing function, oral parafunctions, and symptoms of dysfunction. Thirty-two per cent of participants had at least one dysfunction symptom. Pain on opening was the most common (35.7%), followed by headache (33.6%), and joint sounds (32.2%). Symptoms increased with decline in general health, particularly the health of peripheral joints. Chewing functions were not impaired. Tooth loss did not affect dysfunction symptoms in adolescents, whilst a weak relation was evident between oral parafunctions and symptoms of mandibular dysfunction.