Immunohistochemical mapping of neurophysins and calcitonin gene-related peptide in the human brainstem and cervical spinal cord

Our study investigates the distribution of neurophysins (Nph), proteins that are part of the precursors for vasopressin and oxytocin, and calcitonin gene-related peptide (CGRP) in the human brainstem by immunohistochemistry. Both peptides were found in discrete regions of the human hindbrain. Nph could be demonstrated exclusively in fibers and punctate perineural varicosities that were travelling within the mesencephalic central gray, substantia nigra, as well as locus coeruleus, medial longitudinal fascicle, raphe, nucleus of the solitary tract, lateral reticular nucleus and area postrema. A few varicosities were seen in the substantia gelatinosa of the spinal trigeminal tract and its continuation into the dorsal horn of the cervical spinal cord. In contrast to these observations. CGRP-immunoreactive fibers were found to be densest in the spinal tract of the trigeminal nerve and the dorsal horn of the spinal cord. In addition, fibers and varicosities could be demonstrated in numerous distinct brain regions, such as locus coeruleus and subcoeruleus, solitary tract, cuneate nucleus, raphe and periaqueductal gray. CGRP-immunoreactivity was also present in perikarya in the ventral horn of the spinal cord, as well as motor nuclei of cranial nerves, i.e., hypoglossal nucleus, ambiguous nucleus. Our results suggest that Nph-immunoreactivity in the human brainstem may be present predominantly within long fiber projections from hypothalamic neurosecretory nuclei, in analogy to data obtained from rodents, whereas CGRP may play a role in the branchiomotor system as well as in intrinsic or extrinsic projections involved in autonomic regulation and integration of sensory information.     

Immunohistochemical localization of atrial natriuretic peptide in the developing and adult mammalian kidney

The discovery, within the last decade, of atrial natriuretic peptide (ANP), a family of peptides with natriuretic/diuretic and vasorelaxant properties, has prompted much research into the mechanisms and sites of action of ANP within the kidney. In the present study, ANP was localized in the kidneys of several mammalian species by immunohistochemical techniques (1) to identify possible sites of synthesis; (2) to compare the localization of ANP to known physiological effects; (3) to determine species differences, if any, in ANP localization; and (4) to study the development of ANP immunoreactivity in the fetal and neonatal rat kidney. Using an antibody against rat ANP IV, ANP was localized exclusively on the proximal convoluted tubule (PCT) brush border and within intercalated cells of the outer medullary and cortical collecting tubules and ducts of adult mouse, rat, pig, monkey, and human kidneys. The development of ANP immunoreactivity paralleled the differentiation and maturation of collecting duct epithelium in rat fetal kidney. Atrial natriuretic peptide found within intercalated cells of the cortical and outer medullary collecting ducts may be the result of endogenous synthesis and, following secretion, may be available to receptors in the inner medullary collecting ducts.     

Pneumocystis infection in macaque monkeys: Macaca fuscata fuscata and Macaca fascicularis

Retrospective examination of lungs from 128 monkey necropsies was attempted for Pneumocystis infection using special stains, including toluidine blue-O and Gomori's methenamine silver nitrate. Four Japanese monkeys (7.7%), Macaca fuscata fuscata, and one crab-eating monkey (7.7%), Macaca fascicularis, were found to have Pneumocystis infection. The organism was found in young and infant animals. At the time of death, one infant and two young monkeys were debilitated and/or emaciated. Pneumocystis infection was considered an important lesion which could have caused reduced respiratory function in two of the Japanese monkeys, but constituted only an incidental finding in the others.Contribution No. 567 from the Department of Medical Zoology, Kyoto Prefectural University of Medicine     

Mapping of FGF1 in the Medulla Oblongata of Macaca fascicularis

FGF1 is highly expressed in neurons and it has been proposed to play a role in the neuroprotection and in regeneration. Low FGF1 expression in neurons has been linked to increased vulnerability in cholinergic neurons. Previous reports have shown that the expression of FGF1 in rat brain is localized to the cholinergic nuclei of the medulla oblongata, with low ratio of neurons positive for FGF1 in the dorsal motor nucleus of the vagus (DMNV). The role of FGF1 in the primate brain has yet to be clarified. In this study, we mapped FGF1 immunoreactivity in the medulla oblongata of cynomolgus monkey brainstems. Our results demonstrated that FGF1 immunoreactivity follows the pattern of distribution of cholinergic nuclei in the medulla oblongata; with strong localization of FGF1 to cholinergic neurons of the hypoglossal nucleus, the facial nucleus and the nucleus ambiguus. In contrast, the DMNV shows markedly lower FGF1 immunoreactivity. Localization of FGF1 to cholinergic neurons was only observed in the lateral region of the DMNV, with higher immunoreactivity in the rostral ventral-lateral region of the DMNV. These findings are consistent with the distribution of FGF1 immunoreactivity in previous studies of the rat brain.     

Immunohistochemical localization of acid-sensing ion channel 2 (ASIC2) in cutaneous Meissner and Pacinian corpuscles of Macaca fascicularis

Acid-sensing ion channel 2 (ASIC2) is a member of the degenerin/epithelial sodium channel superfamily, presumably involved mechanosensation. Expression of ASIC2 has been detected in mechanosensory neurons as well as in both axons and Schwann-like cells of cutaneous mechanoreceptors. In these studies we analysed expression of ASIC2 in the cutaneous sensory corpuscles of Macaca fascicularis using immunohistochemistry and laser confocal-scanner microscopy. ASIC2 immunoreactivity was detected in both Meissner and Pacinian corpuscles. It was found to co-localize with neuron-specific enolase and RT-97, but not with S100 protein, demonstrating that ASIC2 expression is restricted to axons supplying mechanoreceptors. These results demonstrate for the first time the presence of the protein ASIC2 in cutaneous rapidly adapting low-threshold mechanoreceptors of monkey, suggesting a role of this ion channel in touch sense.     

The nucleus accumbens in monkeys (Macaca fascicularis)

The nucleus accumbens (NA), which receives inputs from limbic structures and projects to the motor system, may be important for the association of reinforcement with action. There are projections to the NA from the amygdala and hippocampus. Discrimination and reversal learning tasks which are known to be disrupted by lesions to these areas in monkeys were given to monkeys with lesions of the NA. Twelve monkeys (Macaca fascicularis) were used in the present study. Six of these received ibotenic acid lesions which resulted in considerable cell loss in the NA; the remaining six acted as controls. The first group of six monkeys were taught a visual discrimination task pre-operatively. Post-operatively, these monkeys were tested on visual and spatial discrimination and reversal tasks. A second group of six monkeys were tested on a motor reversal task. The results indicate that ibotenic acid lesions of the NA transiently impair spatial but not visual reversal learning in monkeys. The NA lesions did not impair a monkey's ability to perform visual or spatial discriminations, or the ability to perform the motor learning or motor reversal tasks. Our results suggest that bilateral lesions of the NA in monkeys do not disrupt the ability to discriminate basic properties of reward-related stimuli or the formation of visual stimulus-reward associations. In addition, our results argue against theories which suggest that the NA is important for behavioural switching or general behavioural flexibility. We conclude that the NA may play a more specific role in the association of temporal and spatial cues with movement and reward.     

Atherosclerosis research in cynomolgus monkeys (Macaca fascicularis)

This is a review of the utilization of cynomolgus monkeys (Macaca fascicularis) in atherosclerosis research. Naturally occurring and experimentally induced atherosclerosis progression and regression studies are described. This species has been utilized as an animal model to study the effects of immunologic injury, aging, exercise, and drug intervention on atherosclerotic lesions. Cynomolgus macaque atherosclerosis induced by feeding cholesterol is a good model of human atherosclerosis because of similar gender-related differences in susceptibility to coronary artery atherosclerosis, a relatively high incidence of myocardial infarction, and characterized psychosocial factors that influence the development of atherosclerosis.     

Distribution of natriuretic peptide receptor-C immunoreactivity in the rat brainstem and its relationship to cholinergic and catecholaminergic neurons

The natriuretic peptide receptor type C (NPR-C) binds all natriuretic peptides. It is thought to be involved in the clearance of natriuretic peptides and more recently has been defined as essential for the neuromodulatory effects of natriuretic peptides. Although the distribution of NPR-C mRNA has been reported in the rat forebrain, there are no data on the distribution of NPR-C in the brainstem. We report an immunofluorescence study on the distribution of NPR-C immunoreactivity in the rat brainstem, and its presence in cholinergic and catecholaminergic neurons. NPR-C immunoreactivity was detected in several regions, including the periaqueductal gray, oculomotor nucleus, red nucleus and trochlear nucleus of the midbrain; the pontine nucleus, dorsal tegmental nucleus, vestibular nucleus, locus coeruleus, trigeminal motor nucleus, nucleus of the trapezoid body, abducens nucleus and facial nucleus of the pons; and the dorsal motor nucleus of the vagus, hypoglossal nucleus, lateral reticular nucleus, nucleus ambiguus and inferior olivary nucleus of the medulla oblongata. Interestingly, NPR-C immunoreactivity was detected in the cholinergic neurons of the oculomotor nucleus, trochlear nucleus, dorsal tegmental nucleus, motor trigeminal nucleus, facial nucleus, dorsal motor nucleus of the vagus, nucleus ambiguus and hypoglossal nucleus. Furthermore, NPR-C immunoreactivity was detected in several catecholaminergic neuronal groups including the A6, A5, A1, C3 and C1 cell groups. These results are consistent with an important role for natriuretic peptides in neuroendocrine regulation and central cardiovascular integration. The extensive distribution of NPR-C in the brainstem supports the hypothesis that NPR-C is involved in the neuromodulatory effect of natriuretic peptides.     

Myocardial hypertrophy in Macaca fascicularis. Structural remodeling of the collagen matrix

The collagen skeleton in the myocardium of 37 non-human primates (Macaca fascicularis) was studied by scanning electron microscopy. Twenty-one hypertensive animals had "pressure-overload" myocardial hypertrophy of 4 to 80 weeks duration induced by unilateral perinephric cellophane insertion and 16 were normotensive controls. The purpose of the study was to examine possible remodeling of the collagen matrix of the hypertrophied pressure overloaded M. fascicularis myocardium. An extensive collagen matrix was demonstrated in nonoperated macaques, consisting of a weave which sequestered groups of myocytes and which was joined to the epimysium by long collagen tendons, and of numerous intermyocyte and myocyte to capillary struts. In addition, strands of collagen were seen crossing interfascicular planes. This collagen skeleton was studied at three periods of perinephritis-induced hypertension with hypertrophy: at 4 weeks (evolutionary); at 32 weeks with mild to moderate hypertension (early compensatory); and at 80 weeks with moderately severe hypertension (late compensatory). These sequential studies revealed that the matrix was remodeled. At 4 weeks there was slight thickening and increase in density of the weave. At 32 weeks, in addition to changes in the weave, the collagen strands became thick and pillar-like and the tendons increased in numbers and thickness, and at 80 weeks some myocytes became encased in collagen. The geometrical arrangement of the normal collagen matrix of the myocardium is similar to series and parallel elastic elements with respect to myocytes, and, accordingly, it may be responsible for the mechanical properties of the myocardium during diastole and systole. The structural alterations in the collagen skeleton which we observed in hypertrophy may play a significant role in deleteriously affecting ventricular function of the pressure-overloaded myocardium.     

Macaca mulatta, fascicularis and nemestrina in AIDS vaccine development

Over the past 20 years, many efforts have been made to develop a vaccine against AIDS. The lack of an animal model that can be productively infected with HIV-1 has been partially replaced by macaque species infected with SIV or chimeric SHIV. Natural SIV and chimeric SHIV cause an infection resembling human AIDS, and Asian monkeys of genus Macaca (species mulatta, fascicularis and nemestrina) should be considered a useful surrogate in vaccine trials. A multitude of vaccines and immunization approaches have been evaluated, including live-attenuated viruses, DNA vaccines, subunit proteins and viral and bacterial vectors. The results of all these studies are often difficult to interpret due to lack of standardizations, choice of challenging virus and differences in the macaque species used. This article aims at summarizing the main characteristics of the three macaque species used in vaccine trials.     

Epidemic typhus infection in cynomolgus monkeys (Macaca fascicularis).

A nonhuman primate model of clinical Rickettsia prowazekii infections was developed in cynomolgus monkeys (Macaca fascicularis). Monkeys infected intravenously with 10(7) plaque-forming units developed clinical signs of illness and pathological changes characteristic of epidemic typhus infection in humans. Increases in total leukocyte counts, serum alkaline phosphatase, blood urea nitrogen, and serum glutamic pyruvate transaminase values were observed. Microscopic examination revealed typical typhus nodules in the brains of two monkeys that died. These data indicated that the cynomolgus monkey is a suitable model for study of the pathogenesis of epidemic typhus infection and may prove valuable in the evaluation of candidate R. prowazekii vaccines.     

Carbonic anhydrase in epithelia and fenestrated juxtaepithelial capillaries of Macaca fascicularis

Thirty-nine epithelia of the monkey Macaca fascicularis and their underlying connective tissue were stained for carbonic anhydrase (CA) and studied by light microscopy. The capillaries immediately adjacent to the epithelium, 'juxtaepithelial', were also studied by electron microscopy with special attention to presence or absence of fenestrations. Only juxtaepithelial capillaries stained. These were fenestrated under many epithelia and all fenestrated juxtaepithelial capillaries stained. Often staining and fenestration were limited to the part of their circumference facing the epithelium. There was a strong association P less than 0.002 between CA-staining and fenestration of the capillaries, a moderately strong association P less than 0.02 between CA-staining of the membrane of the epithelium and the adjacent capillaries, and no association between staining of the epithelial cytoplasm on the one hand and the epithelial membrane or the capillaries on the other. Fenestrated capillaries containing CA despite unstained epithelia were found in the thyroid, parathyroid, adrenal cortex, islets and anterior pituitary, and also, but not consistently, in the prostatic and the seminal glands. Unfenestrated capillaries containing CA were found in the stomach, colon and lung, and in the Graffian follicle and Fallopian tube. The hypothesis is advanced that capillary CA besides conventional functions plays a part in conserving the state of fenestration of the endothelium and that CA-inhibitors could exert part of their therapeutic and side actions by reducing fenestrations.     

Characterization of Meibomian gland innervation in the cynomolgus monkey (Macaca fascicularis)

To characterize the innervation of the cynomolgus monkey (Macaca fascicularis) Meibomian (tarsal) glands, upper lids of six cynomolgus monkeys were investigated with electronmicroscopical and double-labeling immunocytochemical methods. Antibodies against calcitonin gene-related peptide (CGRP), dopamine-β-hydroxylase (DBH), neuropeptide Y (NPY), nitric oxide synthase (NOS), protein gene product 9.5 (PGP 9.5), substance P (SP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) were used. In addition, sections were processed for NADPH-diaphorase (NADPH-d) histochemistry. Staining for PGP 9.5 and electron microscopy showed that Meibomian gland acini were surrounded by a network of unmyelinated nerves and terminal varicose axons. The terminals contained small agranular (30–60 nm) and large granular vesicles (65–110 nm), and were observed in close contact with the basal lamina of the acini, but never internally to the basal lamina. Meibomian axons showed like-immunore-activity (LI) for the neuropeptides SP, CGRP, NPY, and VIP. In addition, the axons stained for TH, DBH, NOS, and NADPH-d. VIP-LI, NOS- and NADPH-d-positive axons appeared to be more numerous, TH- and DBH-positive axons more rare than others. Most SP-LI axons were double-labelled for CGRP-LI, some for VIP-LI or NPY-LI. In addition, some VIP-LI axons were double-labeled for NPY-LI. NPY/VIP-LI and NPY/SP-LI axons were only observed close to the Meibomian acini. Conversely, NPY-LI colocalized with TH-IR or DBH-IR predominated in perivascular nerves of Meibomian gland vasculature. The close association of varicose axons with the acini of Meibomian glands indicates that nervous signals modulate meibomian secretion. Meibomian gland nerve fibers in the cynomolgus monkey appear to utilize various neuropeptides, catecholamines and nitric oxide as transmitter substances, and seem to derive from the pterygopalatine, superior cervical and trigeminal ganglion respectively.     

Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis)

Anthrax is considered a serious biowarfare and bioterrorism threat because of its high lethality, especially by the inhalation route. Rhesus macaques (Macaca mulatta) are the most commonly used nonhuman primate model of human inhalation anthrax exposure. The nonavailability of rhesus macaques necessitated development of an alternate model for vaccine testing and immunologic studies. This report describes the median lethal dose (LD(50)) and pathology of inhalation anthrax in cynomolgus macaques (Macaca fascicularis). Gross and microscopic tissue changes were reviewed in 14 cynomolgus monkeys that died or were killed after aerosol exposure of spores of Bacillus anthracis (Ames strain). The LD(50) and 95% confidence intervals were 61800 (34000 to 110000) colony-forming units. The most common gross lesions were mild splenomegaly, lymph node enlargement, and hemorrhages in various organs, particularly involving the meninges and the lungs. Mediastinitis, manifested as hemorrhage or edema, affected 29% of the monkeys. Microscopically, lymphocytolysis occurred in the intrathoracic lymph nodes and spleens of all animals, and was particularly severe in the spleen and in germinal centers of lymph nodes. Hemorrhages were common in lungs, bronchial lymph nodes, meninges, gastrointestinal tract, and mediastinum. These results demonstrate that the Ames strain of B. anthracis is lethal by the inhalation route in the cynomolgus macaque. The LD(50) of the Ames strain of B. anthracis was within the expected experimental range of previously reported values in the rhesus monkey in an aerosol challenge. The gross and microscopic pathology of inhalation anthrax in the cynomolgus monkey is remarkably similar to that reported in rhesus monkeys and humans. The results of this study are important for the establishment of an alternative nonhuman primate model for evaluation of medical countermeasures against inhalational anthrax.     

Features of cortically evoked swallowing in the awake primate (Macaca fascicularis).

Although the cerebral cortex has been implicated in the control of swallowing, the output organization of the cortical swallowing representation, and features of cortically evoked swallowing, remain unclear. The present study defined the output features of the primate "cortical swallowing representation" with intracortical microstimulation (ICMS) applied within the lateral sensorimotor cortex. In four hemispheres of two awake monkeys, microelectrode penetrations were made at 5 mm deep to the cortical surface corresponding to both the white matter underlying the CMA and the frontal operculum; EMG patterns of swallows elicited from these four cortical regions showed some statistically significant differences. Whereas swallowing ONLY was evoked at some sites, particularly within the deep cortical area, swallowing was more frequently evoked together with other orofacial responses including rhythmic jaw movements. Increasing ICMS intensity increased the magnitude, and decreased the latency, of the swallow-related EMG burst in the genioglossus muscle at some sites. These findings suggest that a number of distinct cortical foci may participate in the initiation and modulation of the swallowing synergy as well as in integrating the swallow within the masticatory sequence.     

Some topographical connections of the striate cortex with subcortical structures in Macaca fascicularis

Summary Subcortical connections of the striate cortex with the superior colliculus (SC), the lateral pulvinar (Pl), the inferior pulvinar (Pi) and the dorsal lateral geniculate nucleus (LG) were studied in the macaque monkey, Macaca fascicularis, following cortical injections of tritiated proline and/or horseradish peroxidase. All four structures were shown to receive topographically organized projections from the striate cortex. The exposed surface of the striate cortex was found to be connected to the rostral part of the SC and the caudal part of the LG. Injections of the exposed striate cortex close to its rostral border resulted in label in adjoining parts of the Pl and Pi. The ventral half and dorsal half of the calcarine fissure were connected with the medial and lateral parts of the SC, the ventrolateral and dorsomedial portions of the Pl and Pi and the lateral and medial parts of the LG, respectively. Injections located at the lateral posterior extreme of the calcarine fissure resulted in label at the optic disc representation in the LG. The horseradish peroxidase material demonstrated that LG neurons in all laminae and interlaminar zones project to the striate cortex.Abbreviations BIC brachium of the inferior colliculus - BSC brachium of the superior colliculus - C cerebellum - CG central grey - i interlaminar zone(s) of the dorsal lateral geniculate nucleus - IC inferior colliculus - ICc central nucleus of the inferior colliculus - LG dorsal lateral geniculate nucleus - m magnocellular layer(s) of the dorsal lateral geniculate nucleus - MG medial geniculate body - p parvocellular layer(s) of the dorsal lateral geniculate nucleus - P pulvinar complex - Pi inferior pulvinar - PG pregeniculate nucleus - Pl lateral pulvinar - Pm medial pulvinar - s superficial layer(s) of the dorsal lateral geniculate nucleus - SC superior colliculus - sgs stratum griseum superficiale of the superior colliculus - R reticular nucleus of the thalamus - VP ventroposterior group - 17 Area 17 Supported by NEI Grants EY-07007 (J. Graham) and EY-02686 (J.H. Kaas)     

Experimental African HEV infection in cynomolgus macaques (Macaca fascicularis)

Experimental infection with hepatitis E virus (HEV) from Africa has not been investigated. Our purpose was to study hepatitis E produced by HEV from Chad (North Africa) and to analyze the genetic sequence of the HEV obtained after animal passage. An HEV-containing fecal sample from Chad was intravenously inoculated in four cynomolgus macaques. When serum Alanine Amino Transferase (ALT) levels rose, open liver biopsy and bile aspiration were performed. In all the monkeys, an ALT rise occured 25 to 32 days after inoculation and new anti-HEV was detected by Enzyme Immuno Assay (EIA). Hepatic histopathology was consistent with acute viral hepatitis. HEV was detected by polymerase chain reaction (PCR) in bile (3/4 animals) and feces (2/4 animals) and by imunoelectron microscopy (IEM) in the inoculum and one bile specimen. A genetic variant HEV was identified in one monkey. The Chad HEV produced hepatitis E with pathophysiologic and histopathologic findings similar to those observed with HEV from other geographic origins. A genomic variant HEV population was produced after one passage in a macaque. J. Med. Virol. 55:197–202, 1998. © 1998 Wiley-Liss, Inc.     

Chronic lymphocytic thyroiditis in a cynomolgus macaque (Macaca fascicularis)

Chronic lymphocytic thyroiditis characterized by multifocal follicular lymphoid cell infiltrates with germinal centers, thyroid acinar atrophy and pituitary cell hyperplasia/hypertrophy of the adenohypophysis was detected in a vehicle control, 4-year-old female Cynomolgus macaque in a routine toxicology study. Lymphoid cells of germinal centers were positive for the B-cell marker CD20 by immunohistochemistry (IHC), while remaining lymphocytes were positive for the T-cell marker CD3. Hypertrophied/hyperplastic pituitary cells were positive for thyroid stimulating hormone (TSH) by IHC, consistent with an adaptive response due to removal of hormonal negative feedback from the diseased thyroid gland. Features of this case are similar to chronic lymphocytic thyroiditis in humans, an autoimmune disorder also known as Hashimoto's disease. Chronic lymphocytic thyroiditis with compensatory pituitary changes may occur spontaneously in young, clinically normal cynomolgus macaques and its presence in drug treated animals should be interpreted with caution.