Marginal analysis of multiple outcomes with informative cluster size.

In surveillance studies of periodontal disease, the relationship between disease and other health and socioeconomic conditions is of key interest. To determine whether a patient has periodontal disease, multiple clinical measurements (eg, clinical attachment loss, alveolar bone loss, and tooth mobility) are taken at the tooth-level. Researchers often create a composite outcome from these measurements or analyze each outcome separately. Moreover, patients have varying number of teeth, with those who are more prone to the disease having fewer teeth compared to those with good oral health. Such dependence between the outcome of interest and cluster size (number of teeth) is called informative cluster size and results obtained from fitting conventional marginal models can be biased. We propose a novel method to jointly analyze multiple correlated binary outcomes for clustered data with informative cluster size using the class of generalized estimating equations (GEE) with cluster-specific weights. We compare our proposed multivariate outcome cluster-weighted GEE results to those from the convectional GEE using the baseline data from Veterans Affairs Dental Longitudinal Study. In an extensive simulation study, we show that our proposed method yields estimates with minimal relative biases and excellent coverage probabilities.

Five-year clinical results for treatment of intrabony defects with EMD, guided tissue regeneration and open-flap debridement: a case series.

BACKGROUND AND OBJECTIVE: Although regenerative periodontal surgery with EMD or guided tissue regeneration (GTR) has been shown to enhance periodontal regeneration, there are limited data on the long-term results following these treatment modalities. The purpose of the present study was to investigate the long-term clinical outcomes in intrabony defects following regenerative periodontal surgery with EMD or GTR compared with open-flap debridement (OFD). MATERIAL AND METHODS: Data from 40 subjects (44 teeth), with no history of smoking or systemic diseases that could interfere with periodontal disease and who received one of three surgical procedures (EMD, GTR or OFD) for two- or three-wall intrabony defects, were analyzed. Postoperative reduction in probing pocket depth, gain in clinical attachment level, gingival recession and percentage bone fill were compared at 1, 3 and 5 years. RESULTS: Reduction in probing pocket depth after GTR was significantly higher than after OFD at 1 and 3 years postoperatively, but there was no difference between the groups at 5 years. The gains in clinical attachment level for EMD (at 3 and 5 years) and for GTR (at 1, 3 and 5 years) were significantly greater than for OFD. Gingival recession after treatment with EMD and GTR showed a tendency toward positive results, whereas no such tendency was observed for OFD. Postoperative percentage bone fill for EMD and GTR was significantly greater than for OFD at 3 and 5 years. CONCLUSIONS: This is a retrospective study and an exploratory report with a high risk of bias. Within the limits of the current study, it may be concluded that superior gains in clinical attachment level and improved percentage bone fill can be obtained with EMD and GTR when compared with OFD, and these can be maintained over a period of 5 years.

Inflammatory bone loss in experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in interleukin-1 receptor antagonist knockout mice.

The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.

Follicular dendritic cell-secreted protein is decreased in experimental periodontitis concurrently with the increase of interleukin-17 expression and the Rankl/Opg mRNA ratio.

BACKGROUND AND OBJECTIVE: T-helper type 17 (Th17) cells produce interleukin-17 (IL-17) and help to protect against inflammation and infection in periodontal disease. Furthermore, while follicular dendritic cell-secreted protein (FDC-SP) may be involved in the inflammation of periodontal tissue, the biological role of FDP-SP in periodontal disease is still unknown. The purpose of the present study was to clarify the expression of IL-17 and FDC-SP in experimental periodontitis in rats. MATERIAL AND METHODS: Seven-week-old male Wistar rats were divided into baseline control, sham and test groups. Experimental periodontitis was induced by placing a ligature in the mesiopalatal area, and untreated rats served as a baseline control group. Morphological changes in alveolar bone were investigated 7, 14 and 28 d after treatment. Expression of the Rankl, osteoprotegerin (Opg) and Il17 genes was analyzed 5 and 7 d after the induction of experimental periodontitis. RESULTS: Alveolar bone resorption progressed in the test group for 7 d, but not thereafter. At 5 d after the induction of periodontitis, the Rankl/Opg mRNA ratio and the expression of IL-17 in the test group were significantly increased compared with the respective values in the baseline control group; however, there were no significant differences between the test and control groups at 7 d. The expression of FDC-SP was significantly decreased in the test group compared with the baseline control group at 5 and 7 d after the induction of periodontitis, and this value had returned to normal levels at 14 and 28 d. CONCLUSION: These results suggest that both IL-17 and FDC-SP could be involved in the inflammatory response, and FDC-SP in the junctional epithelium might play an important role in the Th17 cell-related immune response.

Antimicrobial photodynamic therapy using a diode laser with a potential new photosensitizer, indocyanine green-loaded nanospheres, may be effective for the clearance of Porphyromonas gingivalis.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) is a new treatment method for the removal of infectious pathogens using a photosensitizer and light of a specific wavelength, e.g., toluidine blue with a wavelength of about 600 nm. We explored a new photosensitizer and focused on indocyanine green (ICG), which has high absorption at a wavelength of 800-805 nm. We investigated the bactericidal effect of PDT on Porphyromonas gingivalis using a new photosensitizer, ICG-loaded nanospheres with an 805 nm wavelength low-level diode laser irradiation. METHODS: We designed ICG-loaded nanospheres coated with chitosan (ICG-Nano/c) as a photosensitizer. A solution containing Porphyromonas gingivalis (10(8)  CFU/mL) with or without ICG-Nano/c (or ICG) was prepared and irradiated with a diode laser or without laser irradiation as a negative control. The irradiation settings were 0.5 W with a duty ratio of 10%, for 3-100 ms in repeated pulse (RPT) or continuous wave mode. CFU were counted after 7 d of anaerobic culture. RESULTS: We observed that ICG-Nano/c could adhere to the surface of P. gingivalis. When ICG-Nano/c was used for aPDT, irradiation with RPT 100 ms mode gave the lowest increase in temperature. Laser irradiation with ICG-Nano/c significantly reduced the number of P. gingivalis (i.e., approximately 2-log10 bacterial killing). The greatest bactericidal effect was found in the RPT 100 ms group. However, laser irradiation (RPT 100 ms) with ICG, as well as without photosensitizer, had no effect on the number of bacteria. CONCLUSIONS: Within the limits of this study, ICG-Nano/c with low-level diode laser (0.5 W; 805 nm) irradiation showed an aPDT-like effect, which might be useful for a potential photodynamic periodontal therapy.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up-regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin-1 receptor antagonist-deficient mice.

BACKGROUND AND OBJECTIVE: The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS: Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 µg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. RESULTS: The levels of IL-1α, IL-1ß, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice. CONCLUSION: IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.

Role of Mfa5 in Expression of Mfa1 Fimbriae in Porphyromonas gingivalis.

Fimbriae are protein-based filamentous appendages that protrude from the bacterial cell surface and facilitate host adhesion. Two types of fimbriae, FimA and Mfa1, of the periodontal pathogen Porphyromonas gingivalis are responsible for adherence to other bacteria and to host cells in the oral cavity. Both fimbrial forms are composed of 5 proteins, but there is limited information about their polymerization mechanisms. Here, the authors evaluated the function of Mfa5, one of the Mfa1 fimbrial accessory proteins. Using mfa5 gene disruption and complementation studies, the authors revealed that Mfa5 affects the incorporation of other accessory proteins, Mfa3 and Mfa4, into fibers and the expression of fimbriae on the cell surface. Mfa5 is predicted to have a C-terminal domain (CTD) that uses the type IX secretion system (T9SS), which is limited to this organism and related Bacteroidetes species, for translocation across the outer membrane. To determine the relationship between the putative Mfa5 CTD and the T9SS, mutants were constructed with in-frame deletion of the CTD and deletion of porU, a C-terminal signal peptidase linked to T9SS-mediated secretion. The ∆CTD-expressing strain presented a similar phenotype to the mfa5 disruption mutant with reduced expression of fimbriae lacking all accessory proteins. The ∆porU mutants and the ∆CTD-expressing strain showed intracellular accumulation of Mfa5. These results indicate that Mfa5 function requires T9SS-mediated translocation across the outer membrane, which is dependent on the CTD, and subsequent incorporation into fibers. These findings suggest the presence of a novel polymerization mechanism of the P. gingivalis fimbriae.

Effects of diltiazem and noradrenaline on extracellular potassium changes in the globally ischaemic rat heart.

OBJECTIVE: The aim was to investigate the effects of a calcium antagonist (diltiazem) and a catecholamine (noradrenaline) on extracellular potassium accumulation during global ischaemia. METHODS: Extracellular potassium concentration ([K+]e) was measured during 30 min global ischaemia in the isolated rat heart using a valinomycin potassium sensitive electrode. Contracture development during ischaemia was measured throughout with an intraventricular balloon inserted into the left ventricle and myocardial adenine nucleotides were measured in separate series of hearts. RESULTS: In control hearts, [K+]e showed a characteristic triphasic change during 30 min global ischaemia. This consisted of an early rising phase followed by a transient falling phase after the initial peak of [K+]e, and then a late rising phase. Diltiazem suppressed the rate of rise of [K+]e during early ischaemia, but extended the time course of the early [K+]e rise with the higher dose, abolishing the transient falling phase of [K+]e. During late ischaemia, the rise in [K+]e was attenuated by diltiazem. Noradrenaline also suppressed the early extracellular potassium accumulation, but in contrast to diltiazem, hastened the time course of the late [K+]e rise. CONCLUSIONS: Although diltiazem suppresses the early potassium loss during ischaemia as previously described, the drug also decreases the [K+]e fall by some as yet unknown mechanism, so that the [K+]e level becomes higher than control during the falling phase.

Effects of propentofylline on hypoxia-hypoglycemia-induced calcium accumulation in gerbil hippocampal slices.

The xanthine derivative propentofylline (HWA 285) has been reported to show protective effects against neuronal damage induced by cerebral ischemia. In the present study, microfluorometry was used to investigate the effect of propentofylline on the hypoxia-hypoglycemia-induced intracellular calcium accumulation in gerbil hippocampal slices. When slices were superfused with hypoxic-hypoglycemic medium that did not contain propentofylline, an acute increase in calcium accumulation was detected 75-200 s (mean latency of 123 s) after the beginning of hypoxia-hypoglycemia. When slices were superfused with hypoxic-hypoglycemic mediums that contained 10 microM, 100 microM, and 1 mM propentofylline, the latency of the acute increase in calcium accumulation was prolonged in all subregions of the hippocampus in a dose-dependent manner: mean latencies in field CA1 were 146, 168, and 197 s after hypoxia-hypoglycemia, respectively. This retardation in calcium accumulation may be involved in the mechanisms by which propentofylline diminishes ischemic injury.

Delayed neuronal death is induced without postischemic hyperexcitability: continuous multiple-unit recording from ischemic CA1 neurons.

It has been proposed that neuronal hyperexcitability during postischemic chronic stage mediates delayed neuronal death in the hippocampal CA1 region. In the present study, multiple-unit spike discharges were continuously recorded from hippocampal CA1 neurons of the awake Mongolian gerbil for 5 days after 5 min of ischemia. Before ischemia, CA1 neurons showed burst-like spike discharges (so-called complex spikes). Spike discharges disappeared 8-40 s after the onset of 5-min ischemia and reappeared 5-30 min after recirculation. The frequency of discharges gradually increased but did not return to the preischemic level. The amplitude of the spike discharges was smaller than that recorded before ischemia and the number of spikes composing the burst-like discharges diminished. CA1 neurons did not show any hyperexcitability for 5 days. However, histological examinations revealed widespread neuronal death in the CA1 region. These results indicate that the delayed neuronal death in the hippocampal CA1 region is induced without postischemic neuronal hyperexcitability.

Postischemic enhancements of N-methyl-D-aspartic acid (NMDA) and non-NMDA receptor-mediated responses in hippocampal CA1 pyramidal neurons.

Glutamate receptor-mediated responses were investigated by using a whole-cell recording and an intracellular calcium ion ([Ca2+]i) imaging in gerbil postischemic hippocampal slices prepared at 1, 3, 6, 9, 12, and 24 hours after 5-minute ischemia. Bath application of N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate showed that NMDA-, AMPA- and kainate-induced currents were enhanced in postischemic CA1 pyramidal neurons at 1 to 12 hours after 5-minute ischemia. NMDA and non-NMDA receptor-mediated excitatory postsynaptic currents (EPSC) were examined in postischemic CA1 pyramidal neurons at 3 hours after 5-minute ischemia to confirm whether synaptic responses are enhanced in the postischemic CA1 pyramidal neurons. The amplitudes of NMDA- and non-NMDA-receptor-mediated EPSC were enhanced in the postischemic CA1 pyramidal neurons. NMDA-, AMPA-, and kainate-induced [Ca2+]i elevations were also examined to determine whether the enhancement of currents is accompanied by the enhancement of [Ca2+]i elevation. The enhancements of NMDA-, AMPA-, and kainate-induced [Ca2+]i elevations were shown in the postischemic CA1. These results indicate that NMDA and non-NMDA receptor-mediated responses are persistently enhanced in the CA1 pyramidal neurons 1 to 12 hours after transient ischemia, and suggest that the enhancement of glutamate receptor-mediated responses may act as one of crucial factors in the pathologic mechanism responsible for leading postischemic CA1 pyramidal neurons to irreversible neuronal injury.

Neuronal connections in the primary auditory cortex: an electrophysiological study in the cat.

Neuronal connections in the primary auditory cortex (AI) of the cat were studied electrophysiologically by using intracellular recording techniques. Fast-conducting fibers from the medial geniculate nucleus (MG) projected monosynaptically onto AI neurons in layers III-VI (mainly in layer IV), whereas slow-conducting MG-fibers projected monosynaptically onto AI neurons in layer I. AI neurons which received monosynaptic inputs from the auditory association cortices (AII and Ep) and/or from the contralateral AI were distributed in all layers of the AI; the commissural fibers from the contralateral AI were divided into fast- and slow-conducting ones. AI neurons were categorized into seven types: type I neurons which received monosynaptic inputs from slow-conducting MG-fibers were located in layer I. Type II neurons which received polysynaptic inputs from the MG were located in layers II-VI. Type III neurons which sent their axons to the AII or Ep were mainly located in layer III. Type IV neurons which sent their axons to the contralateral AI were located mainly in layer III. Type V neurons which received monosynaptic inputs from fast-conducting MG-fibers were located mainly in layer IV. Type VI neurons which projected onto the inferior colliculus were located in the upper part of the layer V. Type VII neurons which projected onto the MG were located in layers V and VI.

Distribution and size of thalamic neurons projecting to layer I of the auditory cortical fields of the cat compared to those projecting to layer IV.

The distribution of thalamocortical neurons projecting to layer I of the cat auditory cortical fields was examined by the horseradish peroxidase (HRP) method. After HRP injection into layer I of the primary auditory cortex (AI), HRP-labeled neuronal cell bodies were distributed mainly in the medial, dorsal, and ventrolateral divisions of the medial geniculate nucleus (MGN) and suprageniculate nucleus (Sg), and additionally in the lateral and medial divisions of the posterior group of the thalamus (Pol and Pom), lateroposterior thalamic nucleus (Lp), and nucleus of the brachium of the inferior colliculus (BIN). After HRP injection into layer I of the second auditory cortex (AII), labeled neurons were seen mainly in the medial, dorsal, and ventrolateral divisions of the MGN and Sg and additionally in the Pom, Lp, and BIN. After HRP injection into layer I of the anterior auditory field (AAF), labeled neurons were located mainly in the medial and dorsal divisions of the MGN, Sg, Pol, and BIN, and additionally in the ventrolateral divisions of the MGN, Pom, and Lp. After HRP injection into layer I of the dorsal part of the posterior ectosylvian gyrus (Epd), labeled neurons were observed chiefly in the medial and dorsal divisions of the MGN, Sg, and Lp and additionally in the ventrolateral division of the MGN, Pom, and BIN. After HRP injection into layer I of the ventral part of the posterior ectosylvian gyrus (Epv), labeled neurons were distributed chiefly in the medial and dorsal divisions of the MGN and Pol and additionally in the ventrolateral division of the MGN, Sg, and BIN. Thus no labeled neurons were found in the ventral division of the MGN after HRP injection into layer I of all auditory cortical fields examined in the present study. The average soma diameters of neurons that were labeled after HRP injection into layer I were statistically smaller than those of neurons that were labeled after HRP injection into layer IV.

Morphological and electrophysiological identification of gigantocellular tegmental field neurons with descending projections in the cat: I. Pons.

Two different descending projections from the pontine gigantocellular tegmental field (PFTG) were defined by the use of intracellular recording and intracellular horseradish peroxidase (HRP) techniques in the cat. Type I neurons (reticulospinal neurons) had antidromic spike potentials produced by stimulation of the ipsilateral medial longitudinal fasciculus (MLF) and sent axons to the ipsilateral MLF. Most type I neurons had large ellipsoidpolygonal somata (mean, 59.7 microns), thick axons (average diameter, 3.33 microns), and slightly oblate large dendritic fields. The mean anteroposterior extent of the dendritic field was 1,492 microns, the mean mediolateral extent was 1,784 microns, and the mean dorsoventral extent was 1,562 microns. There were no type I neurons with axon collaterals. In contrast, type II neurons (reticuloreticular neurons) had antidromic spike potentials produced by stimulation of the bulbar reticular formation (BRF) and sent axons directly to the BRF. In comparison with type I neurons, most type II neurons had smaller ellipsoidpolygonal somata (mean, 40.2 microns), thinner axons (average diameter, 2.32 microns), and smaller, slightly oblate dendritic fields. The mean anteroposterior extent of the dendritic field was 1,264 microns; the mean mediolateral extent was 1,511 microns; and the mean dorsoventral extent was 1,226 microns. Also in contrast to type I neurons, 36% of type II neurons had axon collaterals.

Morphological and electrophysiological identification of gigantocellular tegmental field neurons with descending projections in the cat: II. Bulb.

There are three different descending projections from the bulbar gigantocellular tegmental field (BFTG) in the cat, as defined by intracellular recording and intracellular horseradish peroxidase (HRP) techniques. The first pathway arises from neurons which send axons to the contralateral medial longitudinal fasciculus (cMLF neurons); cMLF neurons show excitatory postsynaptic potentials (EPSPs) after stimulation of the ipsilateral pontine gigantocellular tegmental field (PFTG). Most cMLF neurons have large ellipsoid-polygonal somata (mean, 56.8 micron), thick axons (average diameter, 3.09 microns), mostly non-spiny dendrites and dendritic fields flattened in the anteroposterior direction. No cMLF neurons with axon collaterals in the BFTG are present in the data of this study. The second pathway arises from neurons which send axons to the ipsilateral MLF (iMLF neurons); iMLF neurons show EPSPs after stimulation of the ipsilateral PFTG. Most iMLF neurons have large ellipsoid-polygonal somata (mean, 60.2 microns), thick axons (average diameter, 3.00 microns), mostly non-spiny dendrites and dendritic fields that are only slightly flattened in the anteroposterior direction. As with cMLF neurons, no iMLF neurons with axon collaterals in the BFTG are present in the data of this study. The third pathway arises from neurons that send axons directly into the ipsilateral caudal bulbar reticular formation (iBRF neurons). Most iBRF neurons have smaller ellipsoid-polygonal somata (mean, 38.6 microns), thinner axons (average diameter, 1.84 microns), mostly nonspiny dendrites, and dendritic fields that are flattened in the anteroposterior direction. In contrast to cMLF and iMLF neurons, axon collaterals are present in 73% of iBRF neurons. About half of iBRF neurons have bifurcated axon collaterals with both anterior and posterior projections, and in these neurons antidromic spike potentials are elicited by stimulation of the ipsilateral PFTG.

Descending projections from the gigantocellular tegmental field in the cat: cells of origin and their brainstem and spinal cord trajectories.

The trajectories and the cells of origin of the pontobulbar gigantocellular tegmental field descending pathways were studied in the cat using anterograde WGA-HRP and retrograde HRP techniques. Four main descending pathways and cells of origin were delineated: (1) Predominantly large neurons in the pontine gigantocellular tegmental field (average soma diameter = 43.4 microns) and rostral bulbar gigantocellular tegmental field (41.3 microns) gave rise to reticulospinal fibers descending in the ipsilateral medial longitudinal fasciculus and ventral funiculus and distributed in laminae V-X with an ipsilateral predominance. These were primarily large-diameter fibers. (2) Predominantly large neurons (46.9 microns) in the bulbar gigantocellular tegmental field gave rise to reticulospinal fibers descending in the contralateral medial longitudinal fasciculus and ventral funiculus. These were mainly large-diameter fibers. (3) Neurons of predominantly medium size (29.5 microns) in the pontine gigantocellular tegmental field gave rise to reticuloreticular fibers descending directly to and distributed bilaterally in the bulbar reticular formation. These were small-diameter fibers. (4) Neurons of predominantly medium size (28.9 microns) in the bulbar gigantocellular tegmental field gave rise to reticulospinal fibers descending in the ipsilateral reticular formation and lateral funiculus. These were small-diameter fibers.

Cerebral cortical projections to the reticular regions around the trigeminal motor nucleus in the cat.

Cerebral cortical regions which send projection fibers to the reticular regions around the trigeminal motor nucleus were identified in the cat by the horseradish peroxidase (HRP) method. The reticular region around the trigeminal motor nucleus are known to contain many interneurons for masticatory motoneurons. After injections of HRP into the reticular regions around the trigeminal motor nucleus, HRP-labeled neuronal cell bodies in the cerebral cortex were found in layer V. They were distributed bilaterally in the orbitofrontal cortical regions, mainly in the rostral extension of the orbital gyrus close to the presylvian sulcus; more were located in the floor and lateral bank of the presylvian sulcus than in the crown of the orbital gyrus. After injections of HRP conjugated with wheat germ agglutinin (WGA-HRP) into these cortical regions, many labeled presumed axon terminals were distributed bilaterally in the reticular regions around the trigeminal motor nucleus; mainly in the region ventral to the trigeminal motor nucleus and in the intertrigeminal region between the main sensory trigeminal nucleus and the trigeminal motor nucleus. Terminal labeling in these regions was more prominent after WGA-HRP injection into the lateral bank of the presylvian sulcus than after WGA-HRP injection into the crown of the orbital gyrus. Thus, the present results indicate that the main part of the cortical region projecting directly to the reticular regions around the trigeminal motor nucleus in the cat is folded into the presylvian sulcus.

Hearing loss and glutamate efflux in the perilymph following transient hindbrain ischemia in gerbils.

The mechanism underlying ischemia-induced hearing loss was studied in gerbils with transient hindbrain ischemia. Occlusion of the vertebral arteries caused an increase in the concentration of glutamate in the perilymph and elevated the compound action potential (CAP) threshold to 24.6 dB at 5 minutes. the CAP threshold subsequently recovered on reperfusion, gradually reaching 8.3 dB 120 minutes after reperfusion. Under electron microscopy, afferent dendrites of the cochlear nerve in contact with inner hair cells exhibited abnormal swelling 5 minutes after ischemia/reperfusion. These morphological changes were not observed in cochleas treated with an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-type glutamate receptor antagonist, 6-7-dinitroquinoxaline-2,3-dione (DNQX), before hindbrain ischemia; an N-methyl-D-aspartate (NMDA)-type receptor antagonist, D-2-amino-5-phosphonopentanoate (D-AP5), was ineffective. Moreover, the histopathological alterations noted 5 minutes after reperfusion were spontaneously ameliorated 120 minutes after ischemia/reperfusion. These findings suggest that the ischemia-induced increase in extracellular glutamate concentration with subsequent activation of AMPA/kainate receptors is responsible for neurite degeneration and hearing loss in the early stages following transient hindbrain ischemia.

Morphology and laminar organization of electrophysiologically identified neurons in the primary auditory cortex in the cat.

The morphology of electrophysiologically identified neurons was examined in the primary auditory cortex (AI) of the cat. After stimulation of the medial geniculate nucleus (MG), second auditory cortex, posterior ectosylvian gyrus, contralateral AI, or corpus callosum, intracellular potentials were recorded from AI neurons, which were then injected intracellularly with horseradish peroxidase and recovered. Layer IV neurons, which receive MG fibers monosynaptically, are spiny and nonspiny stellate cells, small and medium-sized nonspiny tufted cells, and fusiform cells. They send their axons to layer III of the AI. Corticocortical AI neurons are medium-sized pyramidal cells in layer III. They receive axons from layer IV neurons of the AI and send their axons to layers I, II, IV, and V of the AI. Horizontal cells in layer I receive slow-conducting MG fibers monosynaptically, and send their axons to layer II of the AI. Stellate cells and small pyramidal cells in layer II receive afferent inputs polysynaptically from the MG. Layer II pyramidal cells receive afferent inputs from the MG via AI neurons in layers I and III, and send their axons to layers V and VI. The axons of layer II stellate cells were distributed within layer II. Pyramidal cells which send their axons to the MG are located in layers V and VI, distributing their axon collaterals to layers III-VI of the AI.

Direct cortical projections to the parabrachial nucleus in the cat.

Direct projections from the cerebral cortex to the parabrachial nucleus in the cat were examined by the horseradish peroxidase (HRP)method. When HRP was injected into the parabrachial nucleus, retrogradely labeled neuronal cell bodies were seen, bilaterally with an ipsilateral predominance, mainly in the orbital gyrus, the lateral bank of the presylvian sulcus, and a restricted region in the infralimbic cortex on the medial surface of the frontal lobe (stereotaxic coordinates; Fr: 22, L: 1, H: -1); all labeled neurons were in deep pyramidal cell layer. After injecting HRP conjugated to wheat germ agglutinin (WGA-HRP) into the cortical regions where retrogradely labeled neurons were found after injecting HRP into the parabrachial nucleus, anterogradely labeled cortical fibers were traced to the parabrachial nucleus. Corticoparabrachial fibers originating from the orbital gyrus and the lateral bank of the presylvian sulcus ran ipsilaterally through the internal capsule and the cerebral peduncle down to the lower brainstem, whereas those from the infralimbic cortex coursed down ipsilaterally through the medial forebrain bundle. These cortical fibers to the parabrachial nucleus were distributed bilaterally with an ipsilateral predominance. Cortical fiber terminals in the parabrachial nucleus were topographically arranged: Corticoparabrachial fibers from the lateral bank of the presylvian sulcus ended most massively in the dorsal part of the lateral parabrachial nucleus. Corticoparabrachial fibers from the orbital gyrus ended most heavily in the medial parabrachial nucleus and less heavily in the lateral parabrachial nucleus. Corticoparabrachial fibers from the infralimbic cortex ended mostly in the parabrachial regions surrounding the brachium conjunctivum.