Congenital coronary artery anomalies in adult population detected using dual source ECG-gated CTA in a single institution.

BACKGROUND: Congenital anomalies of the coronary arteries (CAs) are rare and are often diagnosed incidentally during a conventional coronary angiography. Recently, the incidence of these congenital defects is on the rise particularly after the introduction of the electrocardiography (ECG) gated coronary computed tomographic angiography (CCTA). This innovative radiological screening modality has led to the most precise mapping of the course of the CAs on computed tomographic scan. The aim of the study is to determine the prevalence and describe the CAs congenital anomalies and their variations in Kuwaiti population at a single institution experience. MATERIALS AND METHODS: We analysed the CCTA data obtained consecutively from 842 patients (2013-2014), retrospectively. The inclusion criteria for patients' selection were: atypical chest pain, equivocal ECG, assessment of patency of coronary stents or grafts and pre-operative screening. Information was acquiesced using a dual-source CT scanner with ECG gating. RESULTS: Data analysis revealed that 22 (2.61%) patients were found to have CA anomalies out of the 842 patients who underwent CCTA. Out of these CA anomalies, 13 cases showed more than two ostia, 7 cases showed the ectopic origin of a CA from opposite sinus or non-aortic sinus, 2 cases showed single coronary ostium and 1 case showed coronary artery with pulmonary fistula. Also, myocardial bridging was identified in 78 (9.26%) patients whereas ramus intermedius branch was identified in 160 (19%) patients. CONCLUSIONS: The prevalence of CA anomalies in Kuwait was 2.6%, which is relatively higher than previously reported studies from different countries.

Genistein alleviates testicular ischemia and reperfusion injury-induced spermatogenic damage and oxidative stress by suppressing abnormal testicular matrix metalloproteinase system via the Notch 2/Jagged 1/Hes-1 and caspase-8 pathways.

The aim of the study is to examine the role of matrix metalloproteinases (MMPs) and their inhibitors (TIMP) during testicular ischemia/reperfusion (t I/R). The involvement of the Notch pathway, and their modulation by the antioxidant genistein is also studied. Three groups of male Sprague-Dawley rats were used: sham rats, t I/R rats, and genistein-treated rats (10 mg/kg). The t I/R rat model underwent testicular artery occlusion of the left testis and was subjected to 60 min ischemia followed by 4 h reperfusion. Protein expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured in testicular tissue. Histological examination was performed to assess spermatogenesis. Protein levels of Notch 2, Jagged 1, and hairy/enhancer of split 1 (hes-1) was quantified. The degree of testicular oxidative stress, DNA damage and germ cell apoptosis were also evaluated. T I/R induced severe tubular damage, a significant increase in MMP- 2 and MMP-9 expression and decreased expression TIMP-1 and TIMP-2. Genistein treatment normalized the MMP-TIMP imbalance. Rats subjected to t I/R had low total antioxidant capacity of the testis, decreased superoxide dismutase activity, and increased oxidative DNA damage. Enhanced activities of caspase 8, caspase 3 and PARP were also observed during t I/R. Genistein reversed the t I/R-induced suppression of the Notch 2/Jagged 1/hes-1 pathway. Genistein was also able to salvage the testicular structure and function through restoring the MMP-TIMP anti-proteolytic balance, suppressing spermatogenic damage, alleviating oxidative stress and apoptosis. The Notch pathway is partly involved in inhibiting the t I/R-induced testicular impairment.

Effect of inhibition of the ubiquitin-proteasome-system and IκB kinase on airway inflammation and hyperresponsiveness in a murine model of asthma.

The current treatment of asthma is far from optimal and there is a need for novel therapeutic approaches. NFkB has recently been highlighted as an important pro-inflammatory transcriptional factor and its blockade is believed to represent a new therapeutic approach for asthma. The purpose of this study is to investigate the effects of blocking the actions of NFkB, through inhibition of the ubiquitin-proteasome system (UPS) or IkB kinase (IKK), in a murine model of asthma. Treatment with the UPS inhibitor, MG-132 (0.03 and 0.1 mg/kg), did not significantly affect the ovalbumin-induced increase in total and differential cell numbers, histological changes such as perivascular and peribronchial inflammatory cell infiltration, perivascular and peribronchial fibrosis or the increased Penh to methacholine. In contrast, treatment of mice with the IKK inhibitor, BAY 11-7085, (3 and 10 mg/kg) dose-dependently inhibited the ovalbumin-induced increase in airway leukocyte influx and decreased the percentage of airway lymphocytes, neutrophils and eosinophils. Also, BAY 11-7085-treated (10 mg/kg) mice showed a significant decrease in the histologically assessed inflammatory indices as well as a significant reduction in the ovalbumin-induced increase in Penh to inhaled methacholine. Furthermore, BAY 11-7085 significantly inhibited the ovalbumin-induced increase in the level of phosphorylation of IkBalpha and extracellular regulated kinases (ERK) 1/2, whilst MG-132 significantly increased the phosphorylation of (ERK) 1/2. These findings confirm the critical role that NFkB plays in airway inflammation, highlight the importance of IKK in regulating the pro-inflammatory activity of NFkB and also suggest that UPS may not be a useful drug target for asthma treatment.

A journey across the wall of varicose veins: what physicians do not often see with the naked eye.

OBJECTIVE: To examine varicose veins (VVs) from inside out in order to help surgeons and general practitioners better understand the pathogenesis of the disease and improve their management. MATERIAL AND METHODS: A comprehensive examination of the wall of VVs was performed using transmission electron microscopy. The ultrastructural morphology of the collagen, elastin and smooth muscle content of the wall was analyzed in a sample of 10 patients (4 male and 6 female) and 10 matched controls aged between 37 and 50 years. RESULTS: Analysis of the tunica media revealed that the smooth muscle cells were significantly separated from each other by a marked increase in amorphous and fibrous tissue in which many of the collagen and elastin fibers lost their normal structural arrangement. The cells contained a large number of membrane-bound intracellular vesicles and cytoplasmic vacuoles. The collagen fibers were smaller and thinner than what is commonly seen in normal veins, and they were widely separated from each other. A light electron-lucent center was observed in the middle of the fibers. Similar changes were also seen in the intima and were associated with irregular plaque-like intimal thickening. CONCLUSION: Our study revealed a significant separation among smooth muscle cells in the wall of VVs, and the presence of an abnormal amorphous extracellular matrix and intracytoplasmic vacuoles could reflect 'unusual' possible secretory and phagocytic roles of smooth muscle cells. This could provide an important explanation for the abnormal contractile function of these cells in VVs.

Direct evidence on the immune-mediated spontaneous regression of human cancer: an incentive for pharmaceutical companies to develop a novel anti-cancer vaccine.

To develop an effective pharmaceutical treatment for a disease, we need to fully understand the biological behavior of that disease, especially when dealing with cancer. The current available treatment for cancer may help in lessening the burden of the disease or, on certain occasions, in increasing the survival of the patient. However, a total eradication of cancer remains the researchers' hope. Some of the discoveries in the field of medicine relied on observations of natural events. Among these events is the spontaneous regression of cancer. It has been argued that such regression could be immunologically-mediated, but no direct evidence has been shown to support such an argument. We, hereby, provide compelling evidence that spontaneous cancer regression in humans is immunologically-mediated, hoping that the results from this study would stimulate the pharmaceutical industry to focus more on cancer vaccine immunotherapy. Our results showed that patients with >3 primary melanomas (very rare group among cancer patients) develop significant histopathological spontaneous regression of further melanomas that they could acquire during their life (P=0.0080) as compared to patients with single primary melanoma where the phenomenon of spontaneous regression is absent or minimal. It seems that such regression resulted from the repeated exposure to the tumor which mimics a self-immunization process. Analysis of the regressing tumors revealed heavy infiltration by T lymphocytes as compared to non-regressing tumors (P<0.0001), the predominant of which were T cytotoxic rather than T helper. Mature dendritic cells were also found in significant number (P<0.0001) in the regressing tumors as compared to the non regressing ones, which demonstrate an active involvement of the different arms of the immune system in the multiple primary melanoma patients in the process of tumor regression. Also, MHC expression was significantly higher in the regressing versus the non-regressing tumors (P <0.0001), which reflects a proper tumor antigen expression. Associated with tumor regression was also loss of the melanoma common tumor antigen Melan A/ MART-1 in the multiple primary melanoma patients as compared to the single primary ones (P=0.0041). Furthermore, loss of Melan A/ MART-1 in the regressing tumors significantly correlated with the presence of Melan A/ MART-1-specific CTLs in the peripheral blood of these patients (P=0.03), which adds to the evidence that the phenomenon of regression seen in these patients was immunologically-mediated and tumor-specific. Such correlation was also seen in another rare group of melanoma patients, namely those with occult primary melanoma. The lesson that we could learn from nature in this study is that inducing cancer regression using the different arms of the immune system is possible. Also, developing a novel cancer vaccine is not out of reach.

Post-embedding double-gold labeling immunoelectron microscopic co-localization of neurotransmitters in the rat brain.

BACKGROUND: In this report, we describe a new quantitative electron microscopic protocol based on the use of double colloidal-gold post-embedding immunostaining procedure as markers to analyze the subcellular distribution of enkephalin (ENK) and GABA neurotransmitters simultaneously in the same ultrathin tissue sections in the periaqueductal gray area (PAG) of the rat brain. MATERIAL AND METHODS: Double gold particle signals were significantly improved using a number of technical adjustments for the immunogold electron microscopic co-localization technique of ENK and GABA. The GABA-like neuronal elements were immuno-reacted with 20 nm gold particles and the enkephalin-like immunoreactive neurons were labeled with 10 nm gold particles. This double labeling was more apparent in tissue sections that were deactivated for the gold staining of the first antibody. Excellent double labeling was obtained when we blocked antigenicity of the first antiserum with hot (80 degrees C) paraformaldehyde fumes. To minimize the clumping of the second gold particles around the first gold particles used against the first antibody, we tried different staining order for the neurotransmitters tested in this study. It was necessary to use a detergent (Triton X-100) at very low concentration (0.1%) instead of etching to expose the antigenic determinants of the neurotransmitters and at the same time to reduce the deleterious effects on the morphology of the tissue sections. Furthermore, the high-glutaraldehyde fixation and the decrease in the interval between cutting and labeling of the ultrathin sections significantly improved the results obtained in this study. RESULTS: Double labeling of sections with ENK and GABA produced co-localization in 23.1% and 1.2% of the immunoreactive axonal terminals and dendrites, respectively. Most of the double-labeled terminals contained more GABA-like than ENK-like immunolabeling. Half of the axon terminals [51%] and dendrites [56%] in the ventrolateral PAG were not labeled with either of GABA or ENK immunoreactivity. CONCLUSIONS: This procedure was found to be completely compatible with good double immunolabeling and ultrastructural preservation.

Post-embedding double-gold labeling immunoelectron microscopic co-localization of neurotransmitters in the rat brain.

In this report, we describe a new quantitative electron microscopic protocol based on the use of double colloidal-gold post-embedding immunostaining procedure as markers to analyze the subcellular distribution of enkephalin (ENK) and GABA neurotransmitters simultaneously in the same ultrathin tissue sections in the periaqueductal gray area (PAG) of the rat brain. Double-gold particle signals were significantly improved using a number of technical adjustments for the immunogold electron microscopic co-localization technique of ENK and GABA. The GABA-like neuronal elements were immuno-reacted with 20 nm gold particles and the enkephalin-like immunoreactive neurons were labeled with 10 nm gold particles. This double labeling was more apparent in tissue sections that were deactivated for the gold staining of the first antibody. Excellent double labeling was obtained when we blocked antigenicity of the first antiserum with hot (80 degrees C) paraformaldehyde fumes. To minimize the clumping of the second gold particles around the first gold particles used against the first antibody, we tried different staining order for the neurotransmitters tested in this study. It was necessary to use a detergent (Triton X-100) at very low concentration (0.1%) instead of etching to expose the antigenic determinants of the neurotransmitters and at the same time to reduce the deleterious effects on the morphology of the tissue sections. Furthermore, the high-glutaraldehyde fixation and the decrease in the interval between cutting and labeling of the ultrathin sections significantly improved the results obtained in this study. Double labeling of sections with ENK and GABA produced co-localization in 23.1% and 1.2% of the immunoreactive axonal terminals and dendrites, respectively. Most of the double-labeled terminals contained more GABA-like than ENK-like immunolabeling. Half of the axon terminals [51%] and dendrites [56%] in the ventrolateral PAG were not labeled with either of GABA or ENK immunoreactivity. This procedure was found to be completely compatible with good double immunolabeling and ultrastructural preservation.

Ultrastructural changes in a case of mucoid degeneration of the brachial artery.

An electron microscopic (EM) description of mucoid degeneration of the brachial artery in a 67 year old man is presented. In this case, the affected artery showed mucoid degeneration of the intima and media circumferentially, dissecting and destroying the muscle fibres. Ultrastructurally, mucoid degenerating muscle cells showed numerous large mucin-containing vesicles in the cytoplasm. Cells were widely separated by large accumulation of mucoid material, which appeared to penetrate the extracellular collagen fibre bundles. Most of the nuclei of the smooth muscle cells displayed typical necrotic changes undergoing dissolution or having already broken up into discrete fragments. This case of intimo-medial degeneration (IMMD) suggests that the condition could arise spontaneously anywhere in the inner coats of the arterial system away from the vessels that are close to synovial joints. This is a rare presentation of IMMD of arteries, which has been described mainly in the aorta and its major branches.

Peripheral inflammation is associated with decreased veratridine-induced release of GABA in the rat ventrocaudal periaqueductal gray: microdialysis study.

Systemic administration of opiates or direct injection of opioid peptides into the periaqueductal gray (PAG) produces a profound antinociception which is thought to be associated with inhibition of neuronal activity in the PAG. This inhibitory effect has been postulated to result from opiate inhibition of GABAergic neurons in the PAG. Whether this opioid-GABAergic system is affected in acute pain state has not been investigated. The present study was thus designed to determine the effects of unilateral peripheral inflammation on ventrocaudal PAG gamma-aminobutyric acid (GABA) release in the rat using in vivo microdialysis and subsequent high pressure liquid chromatography (HPLC) analysis. Microdialysis was chosen to perform direct and dynamic studies of amino acid concentrations in the PAG in control rats and in animals subjected to acute and prolonged inflammation caused by injection of 120 microl of Complete Freund's Adjuvant (CFA) into the hind paw. GABA release was significantly decreased in the CFA treated groups both 24 h as well as 7 days post-treatment. GABA release decreased to approximately one-fourth that of the 24 h mineral oil control group. Likewise, veratridine-induced release of GABA was decreased in rats treated with CFA 7 days prior to dialysis. Systemic injection of naloxone (5 mg/kg i.p.) caused selective and significant block in the decrease of veratridine-induced release of GABA in the 24 h CFA-treated rats. Taken together with data from our previous studies, these results suggest that the decrease in veratridine-induced GABA release in this study may be due to an increase opiate inhibition of GABA resulting from the induction of acute or prolonged elevation of nociceptive input.

Mucoid degeneration of the brachial artery: case report and a review of literature.

Cystic adventitial disease (CAD) is a rare cause of intermittent claudication, occurring in approximately 1:1200 claudicants or 1:1000 of those undergoing arteriography. It is most often described in the popliteal artery and is characterised by a mucinous cyst located in the adventitia of the artery, the contents of which resemble those of a ganglion. The origins of adventitial cysts are unknown, but connections to adjacent synovial spaces have been identified, suggesting that the cyst is a variant of a ganglion. In this report, we discuss a rare case of severe mucoid degeneration of the intima and media in a 67-year-old Saudi male patient. The patient presented with a saccular aneurysm of his right "mid-arm" brachial artery and critical ischemia of his right hand from distal embolisation.

Quantitative immunoelectron microscopic colocalization of GABA and enkephalin in the ventrocaudal periaqueductal gray of the rat.

In the present ultrastructural study in the ventrocaudal periaqueductal gray (PAG) of the rat, the relationship and the association between GABAergic and enkephalinergic neuronal elements were investigated using postembedding colocalization immunogold electron microscopic technique in order to establish the precise relationship between these two important neurotransmitters in this part of the brain stem. The GABA-like neuronal elements were immunoreacted with 20 nm gold particles and the enkephalin (ENK)-like immunoreactive neurons were labeled with 10 nm gold particles. Double labeling of sections with ENK and GABA produced colocalization in 23.3% and 1.2% of axon terminals and dendrites, respectively. Most of the double-labeled terminals contained more GABA-like than ENK-like immunolabeling. Approximately 19.4% of the labeled axon terminals and 8.5% of the labeled dendrites contained only GABA-like immunoreactivity, while 24% of the immunolabeled dendrites were immunoreactive with only ENK-like immunoreactivity. The synapses between the two kinds of immunolabeled neuronal profiles appear to be both asymmetrical and symmetrical. GABA-like immunolabeled terminals contained small, clear, pleomorphic or round vesicles and were found to make synapses with ENK-like immunolabeled and nonimmunolabeled dendrites, whereas most of the ENK-like immunolabeled axon terminals contained dense-cored vesicles. Approximately half of the axon terminals (51%) and dendrites (56%) in the ventrolateral PAG were not labeled for either GABA or for ENK immunoreactivity. The results are discussed in terms of GABAergic inhibition of antinociceptive mechanisms in the ventrolateral PAG and of the activation of these mechanisms by ENK neurotransmitter.

Prolonged noxious stimulation increases periaqueductal gray NMDA mRNA expression: a hybridization study using two different rat models for nociception.

The density and distribution of N-methyl-D-aspartate receptor (NMDAR1) mRNA expression in the rat midbrain periaqueductal gray (PAG) following exposure to unilateral peripheral inflammation or chronic constrictive injury (CCI) as models for chronic peripheral nociception were examined using in situ hybridization technique. The NMDAR1 hybridization signal intensities increased significantly in the ventrolateral areas of the caudal and middle thirds of the PAG after 3 days of Complete Freund's Adjuvant (CFA) injection. Likewise, rats subjected to CCI showed significant increase in hybridization signal intensities in comparison to sham-operated animals in both the ipsi- and contra-lateral ventrolateral quadrants of the caudal and middle thirds of the PAG. In the caudal dorsal raphe, the CFA and the CCI treated animals showed a significant increase in signal hybridization compared to control and sham-operated groups while the rostral dorsal raphe showed no significant changes in either CCI or CFA treated groups. In contrast, there was no significant change in signal intensity of NMDAR1 mRNA in the dorsal subdivisions of the PAG following either CCI or CFA treatment. These results demonstrate significant bilateral increase in NMDAR1 mRNA expression in the ventrolateral areas of the caudal and middle thirds of the PAG and the caudal one half of the dorsal raphe following chronic nociception. The up-regulation phenomenon may constitute a reactive mechanism against chronic neuropathic pain in the PAG.

Prolonged noxious stimulation increases periaqueductal gray NMDA mRNA expression: a hybridization study using two different rat models for nociception.

The density and distribution of N-methyl-D-aspartate receptor (NMDAR1) mRNA expression in the rat midbrain Periaqueductal gray (PAG) following exposure to unilateral peripheral inflammation or chronic constrictive injury (CCI) as models for chronic peripheral nociception were examined using the in situ hybridization technique. The NMDAR1 hybridization signal intensities increased significantly in the ventrolateral areas of the caudal and middle thirds of the PAG after 3 days of Complete Freund's Adjuvant (CFA) injection. Likewise, rats subjected to CCI showed significant increases in hybridization signal intensities in comparison to sham operated animals in both the ipsi and contra-lateral ventrolateral quadrants of the caudal and middle thirds of the PAG. In the caudal dorsal raphe, the CFA and the CCI treated animals showed a significant increase in signal hybridization compared to control and sham operated groups while the rostral dorsal raphe showed no significant changes in either CCI or CFA treated groups. In contrast, there was no significant change in signal intensity of NMDAR1 mRNA in the dorsal subdivisions of the PAG following either CCI or CFA treatment. These results demonstrate significant bilateral increase in NMDAR1 mRNA expression in the ventrolateral areas of the caudal and middle thirds of the PAG and the caudal half of the dorsal raphe following chronic nociception. The up-regulation phenomenon may constitute a reactive mechanism against chronic neuropathic pain in the PAG.

Construction of 1 mm microdialysis probe for amino acids dialysis in rats.

We describe here a microdialysis probe with 1 mm opening for precise and confined dialysis area in the awake, freely moving rat. This probe is designed to allow the local diffusion of the perfusion medium to an area approximately 175 microm high, 266 microm wide (mediolateral direction), and 305 microm in rostrocaudal direction. In addition, the probe allows the local application of drugs to the same precise area of interest. The probe was constructed from a piece of 25 gauge tubing with 1 mm hallowed opening located 0.5 mm from the distal (inserting) end. The dialysis fiber which was inserted into the stainless steel 25 gauge tubing and cemented into place has 200 microm diameter and 5000 molecular weight cut off. We tested the probe diffusion extent by direct infusion of fluorogold through the dialysis cannula. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine a sodium channel activator. All amino acids tested showed a significant 80% times decrease in their recovery concentration when compared to their respective concentrations recovered through 2 mm probe constructed earlier in our laboratory (Renno et al., 1992). Tests in awake rats with probes in the ventrocaudal PAG showed stable amounts of 12 different amino acids during repeated (6-8 times) 12 min samples at 3-5 microl/min collecting rate. Depolarization with 75 microM veratridine resulted in significant elevation in extracellular gamma-aminobutyric acid (GABA), aspartate, glutamate, taurine, glycine and citrulline. This design enables us to apply drugs of interest and measure the concentrations of amino acid neurotransmitters to a more precise, delineated and premeasured areas in the CNS.

Microdialysis of excitatory amino acids in the periaqueductal gray of the rat after unilateral peripheral inflammation.

This study measured the release of glutamate (Glu) and aspartate (Asp) amino acid transmitters in the ventrocaudal compartment of the rat periaqueductal gray (PAG) following exposure to unilateral peripheral inflammation. The release of endogenous Glu and Asp from the rat ventrocaudal PAG was monitored with the microdialysis technique in unanesthetized, unrestrained rats. There was significant increase (1,300%) in the basal concentrations of Glu release in the 7 days Complete Freund's Adjuvant (CFA) treated group compared to 24 h mineral oil control group. Amino acid release was induced by infusing veratridine (75 microM, a sodium channel activator) directly through the 1 mm long dialysis probe. Perfusion of veratridine into the ventrocaudal PAG resulted in significant elevation of Glu and Asp amino acids. In the 24 h and 7 days CFA treated rats, veratridine-evoked release of Glu was significantly decreased in the lateral ventrocaudal PAG compared to control rats injected with mineral oil (CFA vehicle). The peak minus baseline concentrations of Glu in 24 h and 7 days CFA treated groups decreased 55.7% and 43.9%, respectively. In contrast, The basal and the peak minus baseline concentrations of Asp showed no significant change between control group and 24 h and 7 days CFA treated animals. The results provide direct evidence that Glu excitatory amino acid may be involved in nociception/nociception modulation pathway in the ventrocaudal PAG.

Light and electron microscopic immunohistochemical localization of N-acetylaspartylglutamate (NAAG) in the olivocerebellar pathway of the rat.

The inferior olive (IO) is the sole contributor of climbing fibers (CF) to the Purkinje cells of the cerebellar cortex. Although the anatomy and the connectivity between the IO and the cerebellum have been well established, there is still controversy regarding the neurotransmitter systems mediating olivocerebellar projections. The excitatory amino acids, glutamate (Glu) and aspartate (Asp), have both been considered as neurotransmitter candidates of olivocerebellar projections in the rat. More recently N-acetylaspartylglutamate (NAAG) has also been proposed as a transmitter of cerebellar climbing fibers based on biochemical and electrophysiological data. The aim of the present study was to determine whether NAAG immunoreactivity is present in the IO and CF at the light and electron microscopic levels and to quantitate the amount of immunogold labeling in olivary neurons and climbing fiber terminals containing this dipeptide. A polyclonal antisera against NAAG was utilized with a peroxidase-labeled avidin-biotin procedure to demonstrate these immunoreactive neurons in the IO at the light microscopic level. Approximately 45% of olivary neurons display NAAG-like immunoreactivity, and their distribution is unevenly clustered throughout the inferior olive. Using postembedding immunogold electron microscopy in combination with quantitative procedures, we found the highest densities of gold particles in the axonal terminals synapsing on olivary neurons (101.0 particles/microns2), in CF terminals (96.3 particles/microns2), and in some mossy fiber terminals (101.0 particles/microns2). Approximately half of the climbing fiber terminals examined were unlabeled. Moderate labeling occurred in CF axons (70.8 particles/microns2), while IO neuronal perikarya were lightly but significantly labeled (41.6 particles/microns2). The localization of NAAG in the subset of cerebellar climbing fiber terminals provides anatomical support for the hypothesis that NAAG may serve as a neurotransmitter/neuromodulator candidate in the olivocerebellar pathway.

Immunocytochemical evidence that quisqualate is selectively internalized into a subset of hippocampal neurons.

Quisqualic acid (QUIS) has been shown to interact with several glutamate receptor subtypes and uptake sites. We have previously demonstrated that a brief exposure of hippocampal cells to QUIS sensitizes them to depolarization by the alpha-amino-omega-phosphonate analogues of glutamate, AP4, AP5, and AP6. This QUIS-induced sensitization is accompanied by the active uptake of QUIS into hippocampal slices. In order to localize the sites of QUIS uptake into rat hippocampal slices, a polyclonal antibody against QUIS was raised in rabbits. Utilizing immunocytochemical techniques, we have identified immunoreactive axons and dendrites after brief exposure times to QUIS, and perikarya after longer exposure times to QUIS. The intensity of the QUIS immunoreactivity increased as the exposure time to QUIS increased. QUIS immunoreactivity was primarily found in stratum oriens and stratum radiatum, of regions CA1, CA2, and CA3 of the hippocampus as well as in the hilus and molecular layer of the dentate gyrus. The distribution and morphology of QUIS immunoreactive cells appeared to be similar to those of GABAergic interneurons. Glial fibrillary acidic protein (GFAP) did not co-localize with the QUIS-internalizing cells suggesting that they are not glia. Ultrastructural analysis revealed QUIS immunoreactive profiles within the stratum radiatum. Immunostained profiles at both the light and EM levels appeared, in many cases, to be swollen and showed signs of degeneration. Such changes were only evident in tissue exposed to QUIS. These data demonstrate that QUIS is taken up by a select group of neurons in the rat hippocampus.

Nitric oxide synthase-containing neural processes on large cerebral arteries and cerebral microvessels.

We studied whether neural processes containing nitric oxide synthase (NOS) are associated with large cerebral arteries and/or intraparenchymal microvessels. The presence of NOS-positive nerves on large cerebral arteries was examined in whole-mount preparations processed for NADPH diaphorase histochemistry, a procedure that stains NOS-containing neurons. The association between NOS-containing neural processes and intracerebral microvessels was studied by electron microscopy in ultrathin brain sections reacted with antibodies against NOS. A dense perivascular plexus of NADPH diaphorase positive axons was observed in the anterior portion of the circle of Willis and its branches while in the basilar artery the innervation was less dense. Lesions of the major sources of perivascular innervation of the cerebral arteries indicated that these nerve fibers arise from the sphenopalatine ganglia. Within the brain parenchyma, NOS immunoreactivity was observed in dendrites and axonal terminals closely associated with the basal lamina of arterioles and capillaries. We conclude that NOS-containing nerves of peripheral origin innervate large cerebral arteries while NOS-containing neural processes of central origin, especially dendrites, are closely associated with cerebral arterioles and capillaries. The presence of NOS in perivascular dendrites raises the possibility that these structures are a major source of NO during neural activity. These findings, collectively, provide morphological evidence supporting the hypothesis that NOS neurons participate in the mechanisms that match neural activity to cerebral blood flow.

Systemic morphine reduces GABA release in the lateral but not the medial portion of the midbrain periaqueductal gray of the rat.

Neuroanatomical, electrophysiological and pharmacological studies have provided indirect evidence indicating that GABAergic neurons play a key role in opiate analgesia mediated by the midbrain periaqueductal gray (PAG) and ventromedial medulla. Although these studies suggest that systemic administration of opiates inhibits GABA release in the PAG, there have been no investigations to date that have directly examined this issue. The present study was thus designed to determine whether systemic morphine injection inhibits GABA release in the PAG of awake, freely moving rats using in vivo microdialysis and subsequent HPLC analysis. Extracellular levels of GABA, glutamate, aspartate, glycine, homocysteic acid and taurine were monitored with the microdialysis technique in either the lateral or medial portion of the ventrocaudal PAG in unanesthetized, unrestrained rats. Amino acid release was induced by infusing veratridine (75 microM, a sodium channel activator) directly through the dialysis probe. The effect of veratridine alone and the effect of veratridine in the presence of systemic morphine on the concentrations of amino acids in the PAG dialysate were determined. There were no significant differences in the basal concentrations of GABA, taurine, aspartate, glutamate, homocysteic acid and glycine between dialysates collected from the medial versus the lateral ventrocaudal PAG. Glycine, taurine and glutamate were present in the highest concentrations in dialysis samples both before and after treatment with veratridine, whereas GABA, homocysteic acid and aspartate were present in the lowest concentrations. Perfusion of veratridine into the ventrocaudal PAG resulted in significant elevation of all amino acids investigated. Except for taurine, no significant difference in veratridine-induced release between the lateral and medial PAG was observed. Tetrodotoxin (TTX) significantly blocked veratridine-induced release of GABA, aspartate, glutamate, glycine and taurine but not homocysteic acid. When rats were injected with morphine (10 mg/kg i.p.), veratridine-induced release of GABA was selectively and significantly decreased in the lateral but not the medial PAG as compared to control rats injected with saline followed by veratridine perfusion. Systemic injection of morphine or saline caused no significant change in the basal concentration of amino acids in PAG dialysate samples. These findings are consistent with the proposed mechanism of action of morphine in the lateral ventrocaudal PAG and offer the first direct evidence that systemic opiates decrease GABA release in this midbrain region.

A quantitative light and electron microscopic analysis of taurine-like immunoreactivity in the dorsal horn of the rat spinal cord.

Taurine has been proposed as an inhibitory neurotransmitter or neuromodulator in the vertebrate central nervous system. Within the spinal cord, taurine has been shown to have a direct inhibitory effect on spinal neurons and to have a selective antinociceptive effect on chemically induced nociception. Although sufficient data exists to suggest that taurine plays a neurotransmitter or neuromodulatory role in the spinal cord, it is not known whether this amino acid is present in axon terminals nor if this amino acid has a unique pattern of distribution within spinal tissue. To address these questions a monoclonal antibody against taurine was employed to localize taurine-like immunoreactivity in the dorsal horn of the rat spinal cord by using both light and electron microscopic techniques. Taurine-like immunoreactivity was most dense and most prominent in laminae I and II of the dorsal horn. A moderate amount of immunoreactivity was also present in laminae VIII and IX and X while the remaining laminae were only lightly stained. In laminae I and II taurine-like immunostaining was evident within neuronal cell bodies, dendrites, myelinated and unmyelinated axons, axon terminals, and astrocytes and their processes. Cell counts of these two laminae indicated that approximately 30% of neuronal perikarya at the C2 level, 52% of neuronal perikarya at the T6 level, and 18% of neuronal perikarya at the L2 level of the cord exhibited taurine-like immunoreactivity. With preembedding diaminobenzidine staining, approximately 20% of the axons examined in laminae I and II were found to be immunoreactive for taurine. Using postembedding immunogold staining in combination with quantitative procedures, the highest densities of gold particles were found in axon terminals containing pleomorphic vesicles and forming symmetrical synapses (36.8 particles/micron2), in a subpopulation of myelinated axons (34.2 particles/micron2), in a subpopulation of neuronal dendrites (32.6 particles/micron2), and in capillary endothelial cells (39.8 particles/micron2). Moderate labeling occurred in astrocytes (20.9 particles/micron2) and neuronal perikarya (18.7 particles/micron2). The localization of taurine to presumptive inhibitory axon terminals provides anatomical support for the hypothesis that taurine may serve an inhibitory neurotransmitter role in the superficial dorsal horn of the spinal cord. On the other hand, its localization to astrocytes and endothelial cells within both the dorsal ventral horns implies that it serves other nonneuronal functions as well.