Structural alterations in fast-spiking GABAergic interneurons in a model of posttraumatic neocortical epileptogenesis.

Electrophysiological experiments in the partial cortical isolation ("undercut" or "UC") model of injury-induced neocortical epileptogenesis have shown alterations in GABAergic synaptic transmission attributable to abnormalities in presynaptic terminals. To determine whether the decreased inhibition was associated with structural abnormalities in GABAergic interneurons, we used immunocytochemical techniques, confocal microscopy and EM in UC and control sensorimotor rat cortex to analyze structural alterations in fast-spiking parvalbumin-containing interneurons and pyramidal (Pyr) cells of layer V. Principle findings were: 1) there were no decreases in counts of parvalbumin (PV)- or GABA-immunoreactive interneurons in UC cortex, however there were significant reductions in expression of VGAT and GAD-65 and -67 in halos of GABAergic terminals around Pyr somata in layer V. 2) Consistent with previous results, somatic size and density of Pyr cells was decreased in infragranular layers of UC cortex. 3) Dendrites of biocytin-filled FS interneurons were significantly decreased in volume. 4) There were decreases in the size and VGAT content of GABAergic boutons in axons of biocytin-filled FS cells in the UC, together with a decrease in colocalization with postsynaptic gephyrin, suggesting a reduction in GABAergic synapses. Quantitative EM of layer V Pyr somata confirmed the reduction in inhibitory synapses. 5) There were marked and lasting reductions in brain derived neurotrophic factor (BDNF)-IR and -mRNA in Pyr cells and decreased TrkB-IR on PV cells in UC cortex. 6) Results lead to the hypothesis that reduction in trophic support by BDNF derived from Pyr cells may contribute to the regressive changes in axonal terminals and dendrites of FS cells in the UC cortex and decreased GABAergic inhibition. SIGNIFICANCE: Injury to cortical structures is a major cause of epilepsy, accounting for about 20% of cases in the general population, with an incidence as high as ~50% among brain-injured personnel in wartime. Loss of GABAergic inhibitory interneurons is a significant pathophysiological factor associated with epileptogenesis following brain trauma and other etiologies. Results of these experiments show that the largest population of cortical interneurons, the parvalbumin-containing fast-spiking (FS) interneurons, are preserved in the partial neocortical isolation model of partial epilepsy. However, axonal terminals of these cells are structurally abnormal, have decreased content of GABA synthetic enzymes and vesicular GABA transporter and make fewer synapses onto pyramidal neurons. These structural abnormalities underlie defects in GABAergic neurotransmission that are a key pathophysiological factor in epileptogenesis found in electrophysiological experiments. BDNF, and its TrkB receptor, key factors for maintenance of interneurons and pyramidal neurons, are decreased in the injured cortex. Results suggest that supplying BDNF to the injured epileptogenic brain may reverse the structural and functional abnormalities in the parvalbumin FS interneurons and provide an antiepileptogenic therapy.

Differential expression of monocyte/macrophage- selective markers in human idiopathic pulmonary fibrosis.

Idiopathic interstitial pneumonias are a group of idiopathic interstitial lung diseases of which idiopathic pulmonary fibrosis (IPF) is the lesion of usual interstitial pneumonia. Although the pathogenic mechanisms remain incompletely understood, disease-specific changes in blood, a readily accessible biospecimen, have not been fully characterized. To identify biomarkers from blood and sera, the immune status of IPF patients and control subjects without structural lung disease was quantified by measuring cell surface markers, mRNA levels, and serum proteins. Statistically significant differences in cellular and molecular markers were observed between the 2 groups. The cytokine receptor IL-17RB was significantly higher in CD14+ peripheral blood mononuclear cells (PBMCs) from IPF patients, whereas expression of the chemokine receptor CXCR4 was lower. Gene expression analyses identified 18 differentially expressed genes out of 195 selected. Of these, EMR1, CCR3, UPAR, FCGR2A, OPN, CEACAM3, CD16a, CD18, CD11b, LTF, and LCN2 were up-regulated, whereas IL-17RB, IL-10, PDGFA, CD301/Clec10a, CD25/IL-2RA, IL-23p19, and IL-15 were down-regulated in IPF. Differentially regulated genes were in the functional areas of inflammation and cell signaling. Serum levels of UPAR and OPN were higher in IPF. These observations reveal significant differences in cell and molecular markers involved in monocyte/macrophage activation and migration, and suggest a role for IL-17RB in IPF.

Epilepsy following cortical injury: cellular and molecular mechanisms as targets for potential prophylaxis.

The sequelae of traumatic brain injury, including posttraumatic epilepsy, represent a major societal problem. Significant resources are required to develop a better understanding of the underlying pathophysiologic mechanisms as targets for potential prophylactic therapies. Posttraumatic epilepsy undoubtedly involves numerous pathogenic factors that develop more or less in parallel. We have highlighted two potential "prime movers": disinhibition and development of new functional excitatory connectivity, which occur in a number of animal models and some forms of epilepsy in humans. Previous experiments have shown that tetrodotoxin (TTX) applied to injured cortex during a critical period early after lesion placement can prevent epileptogenesis in the partial cortical ("undercut") model of posttraumatic epilepsy. Here we show that such treatment markedly attenuates histologic indices of axonal and terminal sprouting and presumably associated aberrant excitatory connectivity. A second finding in the undercut model is a decrease in spontaneous inhibitory events. Current experiments show that this is accompanied by regressive alterations in fast-spiking gamma-aminobutyric acid (GABA)ergic interneurons, including shrinkage of dendrites, marked decreases in axonal length, structural changes in inhibitory boutons, and loss of inhibitory synapses on pyramidal cells. Other data support the hypothesis that these anatomic abnormalities may result from loss of trophic support normally provided to interneurons by brain-derived neurotrophic factor (BDNF). Approaches that prevent these two pathophysiologic mechanisms may offer avenues for prophylaxis for posttraumatic epilepsy. However, major issues such as the role of these processes in functional recovery from injury and the timing of the critical period(s) for application of potential therapies in humans need to be resolved.

Cytoarchitecture of the retinal ganglion cells in the rat.

PURPOSE: To determine the number and cytoarchitecture of retinal ganglion cells (RGCs) in the female Wistar rat, by using a newly devised procedure for rapid RGC counting in the entire retina that avoids assumptions about RGC spatial arrangement. METHODS: RGCs of normal female Wistar rats were retrogradely labeled with a fluorescent tracer. Automated counting was accomplished by applying standard imaging software to analysis of all labeled cells in retinal flatmounts. The method was validated by comparison of automated and manual counts of 70,000 RGCs in frames covering the density range in the normal rat retina of 600 to 3600 RGC/mm(2). RGC numbers were determined for each retina and compared with the contralateral retina of the same animal. RGC density maps were constructed for each retina. RGC size distribution was determined. RESULTS: Automated RGC counting showed a good linear correlation with manual counting (R(2) = 0.9416). Mean total RGC count in 10 rat eyes was 97,609 +/- 3,930 (SEM) per eye. Contralateral eyes differed by an average of 4.1% (3983 plus minus 5098 RGCs). Size analysis calculated from cell areas confirmed that the majority of rat RGCs are between 7 and 21.5 microm in equivalent diameter. The RGC counts for all frames at the same eccentricity in all 10 of the retinas showed that variability increased with eccentricity and increased further as the fractional area of the retina sampled at each eccentricity was reduced. There was also significant variability in the spatial density of the RGCs at the same eccentricity location between different eyes. Comparison of total RGC counts between left and right eyes estimated from RGC counts in sectors of the retina (hemiretinas or quadrants) showed increased variability compared with counting all the RGCs in a retina. CONCLUSIONS: RGCs in the Wistar rat display significant variability in their cytoarchitecture. Such variability can make quantification by sampling problematic for diffuse, and particularly, for focal RGC losses resulting from experimental interventions, unless virtually the entire RGC population is counted.

Quantitative analysis of retinal ganglion cell (RGC) loss in aging DBA/2NNia glaucomatous mice: comparison with RGC loss in aging C57/BL6 mice.

PURPOSE: To quantify the extent and pattern of retinal ganglion cell (RGC) loss in the DBA2/NNia glaucomatous mouse strain as a function of age and compare it with ganglion cell loss in a nonglaucomatous strain. METHODS: All the ganglion cells in retinas of DBA/2NNia and C57/BL6 mice of various ages (five eyes per age group in 3-month intervals from 3 to 18 months of age) were counted. A novel counting method that does not rely on sampling and that uses retrograde labeling of RGCs with Fluorogold (Fluorochrome; Englewood, CO) was used. RGC loss in the glaucomatous DBA/2NNia mouse strain was contrasted to RGC loss in C57 mice at the same ages. The total number of Fluorogold-labeled cells per retina was compared within and among the two strains as a function of age. In addition, RGC density maps were constructed for each retina, and the range of densities for each age group was compared within and among the two strains. IOP in awake, nonsedated DBA/2NNia mice was measured with a rebound tonometer. RESULTS: RGC loss started between 12 and 15 months of age in C57 mice and led to an approximate 46% reduction by 18 months of age. The rate of loss was best approximated by a second-order polynomial curve. In comparison, DBA/2NNia mice also began showing RGC loss at approximately 12 months of age, but it proceeded at a much faster rate, with approximately 64% of their RGCs dying by the 15th month of age but little additional loss thereafter. RGC loss in the DBA animals had a focal pattern that appeared more patchy and showed greater variability than the age-related loss in C57 mice, which was more diffuse. IOP and total retinal area in DBA/2NNia mice began to increase at approximately 6 months of age. IOP normalized after the 12th month of age. CONCLUSIONS: Age-related RGC loss of up to 50% can occur in the C57 mouse by 18 months of age. The loss does not proceed linearly with age, as is often assumed, and differs both in extent and locational pattern from pathologic RGC loss secondary to glaucoma in DBA/2NNia mouse retinas.

Glutamate-induced glutamine synthetase expression in retinal Muller cells after short-term ocular hypertension in the rat.

PURPOSE: To determine the effect of intraocular pressure (IOP) elevation on glutamate-induced expression of glutamine synthetase (GS) in retinal Müller cells of rat eyes. METHODS: Six groups of three rats each were studied. Group I was a normal control group. In Group II, one eye received an intravitreal glutamate injection (75 nmoles) while the contralateral eye served as control. In Groups III and IV, IOP was raised in one eye by episcleral vein cauterization. Moderately elevated IOP was maintained for 1 day in Group III or 1 week in Group IV (35 +/- 1.9-45 +/- 5.2 mm Hg). An additional two groups of rats received bilateral intravitreal glutamate injections (75 nmoles) immediately (Group V), or 6 days (Group VI), after induction of IOP elevation in one eye. One day after glutamate injection the rats in all groups were killed, and the eyes enucleated and fixed. Retinas from left and right eyes of each animal were embedded together in LR White resin (Ted Pella, Redding, CA). Sections were processed for GS immunolabeling with antibodies to GS by two-stage immunogold labeling with silver enhancement. Images of labeled retinas from the two eyes were captured under identical light microscopic conditions and the GS immunoreactivity in Müller cells was compared between the left and right retinas in the same section by image analysis. An additional five rats were included in Group II and the retinas were analyzed by Western blot analysis to confirm immunohistochemical findings. RESULTS: Similar to the finding in the control group (Group I), the GS immunoreactivity of the left and right eyes of Group III and IV remained unchanged even though the right eyes in the two groups had elevation of IOP lasting for 24 hours and 1 week, respectively. However, GS levels were significantly increased by 40 +/- 5.7% in normotensive eyes 24 hours after intravitreal injection of glutamate (Group II). The rise in GS immunoreactivity was abolished in eyes with acute IOP elevation (Group V). In contrast, when the eyes were exposed to high IOP for 1 week (Group VI), the glutamate-induced increase in GS immunoreactivity was restored. CONCLUSIONS: Elevated levels of vitreal glutamate can increase the expression of GS in retinal Müller cells. This increase was blocked if IOP was acutely elevated for 24 hours but was restored if IOP remained elevated for 1 week. This finding suggests that moderate elevation of IOP causes only short-term functional changes of glutamate metabolism (amidation) by retinal Müller cells. However, it is not known to what extent endogenous extracellular glutamate can regulate GS expression in normal eyes or in eyes with glaucoma.

Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

Systemic inflammatory response syndrome (SIRS) is a potentially lethal condition, as it can progress to shock, multi-organ failure, and death. It can be triggered by infection, tissue damage, or hemorrhage. The role of tissue injury in the progression from SIRS to shock is incompletely understood. Here, we show that treatment of mice with concanavalin A (ConA) to induce liver injury triggered a G-CSF-dependent hepatic infiltration of CD11b+Gr-1+Ly6G+Ly6C+ immature myeloid cells that expressed the orphan receptor myeloid DAP12-associated lectin-1 (MDL-1; also known as CLEC5A). Activation of MDL-1 using dengue virus or an agonist MDL-1-specific antibody in the ConA-treated mice resulted in shock. The MDL-1+ cells were pathogenic, and in vivo depletion of MDL-1+ cells provided protection. Triggering MDL-1 on these cells induced production of NO and TNF-α, which were found to be elevated in the serum of treated mice and required for MDL-1-induced shock. Surprisingly, MDL-1-induced NO and TNF-α production required eNOS but not iNOS. Activation of DAP12, DAP10, Syk, PI3K, and Akt was critical for MDL-1-induced shock. In addition, Akt physically interacted with and activated eNOS. Therefore, triggering of MDL-1 on immature myeloid cells and production of NO and TNF-α may play a critical role in the pathogenesis of shock. Targeting the MDL-1/Syk/PI3K/Akt/eNOS pathway represents a potential new therapeutic strategy to prevent the progression of SIRS to shock.

PKC and polyamine modulation of GluR2-deficient AMPA receptors in immature neocortical pyramidal neurons of the rat.

AMPA receptors (AMPARs) mediate the bulk of fast synaptic excitation in the CNS. We have recently shown that AMPAR-dependent synaptic transmission in immature neocortical pyramidal neurons is mediated by GluR2-deficient receptors that can be modulated by intra- or extracellular polyamines (PAs). Phosphorylation of AMPARs, e.g. by PKC, can lead to enhanced excitation, and PAs are known to modulate PKC activity. Therefore, PAs and PKC might interact to influence AMPAR function. To test this hypothesis, we made whole cell recordings from immature (P12-14) layer V pyramidal neurons and assayed two measures of PA influence on synaptic AMPAR function - inward rectification and use-dependent unblock (UDU), with the latter assayed by differences in rectification between a pair of EPSCs evoked at short (50 ms) latencies. We have previously shown that EPSCs in immature pyramidal neurons displayed inward rectification, which was enhanced by intracellular spermine, as was UDU. Staurosporin (ST), a PKC inhibitor, reversed the effect of PA on rectification and UDU, suggesting that PKC modulates postsynaptic activation of AMPARs. Similarly, polyamine-dependent rectification of spontaneous EPSCs was reversed by treatment with ST or GFX109203X, a specific PKC inhibitor. Chelating intracellular Ca(2+) with BAPTA reproduced the effects of ST. In addition, PA immunoreactivity in layer V pyramidal neurons was reduced by PKC inhibition indicating that PKC activity influences PA metabolism. Taken together, these data support the involvement of postsynaptic PKC activation in both the inward rectification and UDU of EPSCs in immature rat cortex, and suggest an important mechanism by which excitatory synaptic transmission can be dynamically modulated by changes in either [Ca(2+)](i) or [PA](i).

Electrophysiological classification of somatostatin-positive interneurons in mouse sensorimotor cortex.

Classification of inhibitory interneurons is critical in determining their role in normal information processing and pathophysiological conditions such as epilepsy. Classification schemes have relied on morphological, physiological, biochemical, and molecular criteria; and clear correlations have been demonstrated between firing patterns and cellular markers such as neuropeptides and calcium-binding proteins. This molecular diversity has allowed generation of transgenic mouse strains in which GFP expression is linked to the expression of one of these markers and presumably a single subtype of neuron. In the GIN mouse (EGFP-expressing Inhibitory Neurons), a subpopulation of somatostatin-containing interneurons in the hippocampus and neocortex is labeled with enhanced green fluorescent protein (EGFP). To optimize the use of the GIN mouse, it is critical to know whether the population of somatostatin-EGFP-expressing interneurons is homogeneous. We performed unsupervised cluster analysis on 46 EGFP-expressing interneurons, based on data obtained from whole cell patch-clamp recordings. Cells were classified according to a number of electrophysiological variables related to spontaneous excitatory postsynaptic currents (sEPSCs), firing behavior, and intrinsic membrane properties. EGFP-expressing interneurons were heterogeneous and at least four subgroups could be distinguished. In addition, multiple discriminant analysis was applied to data collected during whole cell recordings to develop an algorithm for predicting the group membership of newly encountered EGFP-expressing interneurons. Our data are consistent with a heterogeneous population of neurons based on electrophysiological properties and indicate that EGFP expression in the GIN mouse is not restricted to a single class of somatostatin-positive interneuron.

Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model.

Episcleral vein cauterization (EVC) is used in rats to generate a glaucoma model with high intraocular pressure (IOP). The long-term retinal damage in this glaucoma model, however, has not been accurately quantified. We report the location and amount of retinal ganglion cell (RGC) damage caused by (EVC) induced IOP elevation in two rat strains. IOP was raised in one eye of Wistar (N = 5) and Brown-Norway(B-N)(N = 7) rats by EVC and monitored monthly until IOP in contralateral eyes equalized at 5 months post-surgery. Animals were maintained for 3.5-4.5 additional months. B-N rats (N = 7) that had no EVC served as controls for this strain. Scotopic flash ERGs were recorded at baseline and just prior to euthanasia. Automated counts of all retrogradely labeled RGCs in retinal flat-mounts were determined and compared between contralateral eyes. RGC density maps were constructed and RGC size distribution was determined. Oscillatory potentials in the group of eyes which had elevated IOP were decreased at the time of euthanasia, when IOP had returned to normal. The group of normal B-N rats had similar RGC counts between contralateral eyes. In the experimental group the mean number of RGCs was not significantly different between control and experimental eyes, but 1 of 5 Wistar and 2 of 7 B-N experimental eyes had at least 30% fewer RGCs than contralateral control eyes. Total retinal area in B-N experimental eyes was higher compared to contralateral eyes. Cumulative IOP exposure of the experimental eyes was modestly correlated with RGC loss while oscillatory potentials appeared to be inversely related to RGC loss. In retinas with extensive (> 30% RGC loss) but not complete damage, smaller cells were preserved better than larger ones. The above results indicate that RGC loss in both Wistar and B-N strains is variable after a prolonged elevation of IOP via EVC. Such variability despite equivalent IOP levels and ERG abnormalities, suggests unknown factors that can protect IOP-stressed RGCs. Identification and enhancement of such factors could prove useful for glaucoma therapy.

Polyamines modulate AMPA receptor-dependent synaptic responses in immature layer v pyramidal neurons.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) mediate the majority of fast excitation in the CNS. Receptors lacking GluR2 exhibit inward rectification and paired-pulse facilitation (PPF) due to polyamine (PA)-dependent block and unblock, respectively. In this study, we tested whether rectification and PPF in immature, but not mature, pyramidal neurons depend not only on the absence of functional GluR2 but also on the level of endogenous PAs. Whole cell recordings were obtained from layer V pyramidal neurons of P12-P14 or P16-P20 rats in the presence or absence of spermine in the pipette (50 microM). Isolated minimal excitatory synaptic responses were obtained, and paired (20 Hz) stimuli were used to investigate the rectification index (RI) and paired-pulse ratio (PPR). Spermine and its synthetic enzyme, ornithine decarboxylase (ODC), expression was examined using immunostaining and Western blot, respectively. At the immature stage (