Alvin J. Beitz

The organization of afferent projections to the midbrain periaqueductal gray of the rat

The retrograde transport technique was utilized in the present study to investigate the afferent projections to the periaqueductal gray of the rat. Iontophoretic injections of horseradish peroxidase were made into the periaqueductal gray of 22 experimental animals and into regions adjacent to the periaqueductal gray in 6 control animal. Utilization of the retrograde transport method permitted a quantitative analysis of the afferent projections not only to the entire periaqueductal gray, but also to each of its four intrinsic subdivisions. The largest cortical input to this midbrain region arises from areas 24 and 32 in the medial prefrontal cortex. The basal forebrain provides a significant input to the periaqueductal gray and this arises predominantly from the ipsilateral lateral and medial preoptic areas and from the horizontal limb of the diagonal band of Broca. The hypothalamus was found to provide the largest descending input to the central gray. Numerous labeled cells occurred in the ventromedial hypothalamic nucleus, the lateral hypothalamic area, the posterior hypothalamic area, the anterior hypothalamic area, the perifornical nucleus and the area of the tuber cinereum. The largest mesencephalic input to the periaqueductal gray arises from the nucleus cuneiformis and the substantia nigra. The periaqueductal gray was found to have numerous intrinsic connections and contained a significant number of labeled cells both above and below the injection site in each case. Other structures containing significant label in the midbrain and isthmus region included the nucleus subcuneiformis, the ventral tegmental area, the locus coeruleus and the parabrachial nuclei. The medullary and pontine reticular reticular formation provide the largest input to the periaqueductal gray from the lower brain stem. The midline raphe magnus and superior central nucleus also supply a significant fiber projection to the central gray. Both the trigeminal complex and the spinal cord provide a minor input to this region of the midbrain. The sources of afferent projections to the periaqueductal gray are extensive and allow this midbrain region to be influenced by motor, sensory and limbic structures. In addition, evidence is provided which indicates that the four subdivisions of the central gray receive differential projections from the brain stem as well as from higher brain structures.

Bee venom injection into an acupuncture point reduces arthritis associated edema and nociceptive responses

&NA; Bee venom (BV) has traditionally been used in Oriental medicine to relieve pain and to treat inflammatory diseases such as rheumatoid arthritis (RA). While several investigators have evaluated the anti‐inflammatory effect of BV treatment, the anti‐nociceptive effect of BV treatment on inflammatory pain has not been examined. Previous studies in experimental animals suggest that the therapeutic effect of BV on arthritis is dependent on the site of administration. Because of this potential site specificity, the present study was designed to evaluate the anti‐nociceptive effect of BV injections into a specific acupoint (Zusanli) compared to a non‐acupoint in an animal model of chronic arthritis. Subcutaneous BV treatment (1 mg/kg per day) was found to dramatically inhibit paw edema caused by Freunds adjuvant injection. Furthermore, BV therapy significantly reduced arthritis‐induced nociceptive behaviors (i.e. the nociceptive scores for mechanical hyperalgesia and thermal hyperalgesia). These anti‐nociceptive/anti‐inflammatory effects of BV were observed from 12 days through 21 days post‐BV treatment. In addition, BV treatment significantly suppressed adjuvant‐induced Fos expression in the lumbar spinal cord at 3 weeks post‐adjuvant injection. Finally, injection of BV into the Zusanli acupoint resulted in a significantly greater analgesic effect on arthritic pain as compared to BV injection in to a more distant non‐acupoint. The present study demonstrates that BV injection into the Zusanli acupoint has both anti‐inflammatory and anti‐nociceptive effects on Freunds adjuvant‐induced arthritis in rats. These findings raise the possibility that BV acupuncture may be a promising alternative medicine therapy for the long‐term treatment of rheumatoid arthritis.

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.

The distribution of brain-stem and spinal cord nuclei associated with different frequencies of electroacupuncture analgesia

&NA; Immunocytochemical localization of the c‐fos primary gene protein, Fos, was used to identify spinal cord and brain‐stem sites activated by either 4‐Hz or 100‐Hz electroacupuncture (EA) applied to the Zusanli acupuncture points of both hind limbs in lightly anesthetized Sprague‐Dawley rats. The number and distribution of Fos‐immunoreactive neurons in the brain stem and spinal cord of 4‐Hz and 100‐Hz EA‐treated rats were compared with these in anesthesia and room control rats. Compared to non‐stimulated control rats or rats in which EA was applied to a non‐acupuncture point, both 4‐Hz and 100‐Hz EA‐treated groups exhibited a significantly greater number of Fos‐labeled neurons in the dorsal horn of the L2 spinal cord segment, lateral parabrachial nucleus, substantia nigra, nucleus raphe pallidus, dorsal raphe, locus coeruleus, posterior pretectal nucleus, and the lateroventral periaqueductal gray. In the 4‐Hz‐treated group, significant increases in Fos labeling were also observed in the cuneiform nucleus, dorsal and laterodorsal subdivisions of the periaqueductal gray, habenular nucleus, arcuate hypothalamic nucleus, and the lateroventral and lateral hypothalamic nuclei as compared to non‐stimulated controls. The only brain‐stem nucleus that exhibited significantly increased Fos‐immunoreactive neurons in 100‐Hz but not 4‐Hz EA was the rostolateroventral nucleus of the medulla. These results indicate that many brain‐stem regions are activated by both 4‐Hz and 100‐Hz EA but additional brain‐stem regions are selectively activated by 4‐Hz EA which may relate to the opiate sensitivity of 4‐Hz EA. In sum, these data identify several distinct brain‐stem nuclei that may play a role in acupuncture‐mediated analgesia.

The nuclei of origin of brain stem enkephalin and substance P projections to the rodent nucleus raphe magnus

The sites of origin of brain stem enkephalin and substance P projections to the rodent nucleus raphe magnus were studied utilizing the combined horseradish peroxidase retrograde transport-peroxidase-antiperoxidase immunohistochemical technique. Several brain stem areas were found to contain both enkephalin- and substance P-like immunoreactive double labeled neurons following injection of horseradish peroxidase into the raphe magnus. Nuclei providing both enkephalin and substance P inputs to the raphe magnus include the nucleus reticularis paragigantocellularis, the nucleus cuneiformis, the nucleus solitarius and the trigeminal subdivision of the lateral reticular nucleus. Enkephalin projections to the raphe magnus were also found to originate from the dorsal parabrachial nucleus, the nucleus reticularis gigantocellularis pars alpha and from an area which corresponds to the A5 group of Dahlström & Fuxe. Additional neurons containing substance P-like immunoreactivity and horseradish peroxidase reaction product were identified in the superior central raphe nucleus and the nucleus pontis oralis. The midbrain periaqueductal gray contributes very few enkephalin and substance P fibers to the raphe magnus. The nucleus raphe magnus is a key structure in the intrinsic analgesia system and it has also been implicated in other diverse and non-nociceptive functions. The present study identifies several brain stem sites which provide enkephalin and substance P input to this raphe nucleus. Several of these nuclei have been implicated in central analgesic mechanisms or in non-nociceptive autonomic functions. The present investigation raises the possibility that these brain stem regions may modulate neuronal activity in the raphe magnus via enkephalin or substance P projections and thus influence the involvement of the raphe magnus in both opiate related mechanisms of pain control and non-nociceptive functions.

The water-soluble fraction of bee venom produces antinociceptive and anti-inflammatory effects on rheumatoid arthritis in rats.

We recently demonstrated that bee venom (BV) injection into the Zusanli acupoint produced a significantly more potent anti-inflammatory and antinociceptive effect than injection into a non-acupoint in a Freunds adjuvant induced rheumatoid arthritis (RA) model. However, the precise BV constituents responsible for these antinociceptive and/or anti-inflammatory effects are not fully understood. In order to investigate the possible role of the soluble fraction of BV in producing the anti-arthritic actions of BV acupuncture, whole BV was extracted into two fractions according to solubility (a water soluble fraction, BVA and an ethylacetate soluble fraction, BVE) and the BVA fraction was further tested. Subcutaneous BVA injection (0.9 mg/kg/day) into the Zusanli acupoint was found to dramatically inhibit paw edema and radiological change (i.e. new bone proliferation and soft tissue swelling) caused by Freunds adjuvant injection. BVA treatment also reduced the increase in serum interleukin-6 caused by RA induction to levels observed in non-arthritic animals. In addition, BVA therapy significantly reduced arthritis-induced nociceptive behaviors (i.e. nociceptive scores for mechanical hyperalgesia and thermal hyperalgesia). Finally, BVA treatment significantly suppressed adjuvant-induced Fos expression in the lumbar spinal cord at 3 weeks post-adjuvant injection. In contrast, BVE treatment (0.05 mg/kg/day) failed to show any anti-inflammatory or antinociceptive effects on RA. The results of the present study demonstrate that BVA is the effective fraction of whole BV responsible for the antinociception and anti-inflammatory effects of BV acupuncture treatment. Thus it is recommended that this fraction of BV be used for long-term treatment of RA-induced pain and inflammation. However, further study is necessary to clarify which constituents of the BVA fraction are directly responsible for these anti-arthritis effects.

Nitric oxide synthase immunoreactive neurons anatomically define a longitudinal dorsolateral column within the midbrain periaqueductal gray of the rat: analysis using laser confocal microscopy.

Nitric oxide has recently been proposed as a neuronal messenger in both the central and peripheral nervous system. Antibodies against nitric oxide synthase (NOS), the synthesizing enzyme for nitric oxide, were used in combination with immunocytochemistry and confocal laser microscopy to analyze the distribution of this enzyme in the midbrain periaqueductal gray (PAG) of the rat. NOS immunoreactive neurons were localized predominantly in a longitudinally oriented column in the dorsolateral PAG. NOS immunoreactive fibers and processes were scattered throughout the PAG but were most prevalent in the dorsolateral column and in the juxta-aqueductal column. This study provides neurochemical support for the existence of longitudinal columns in the PAG which are postulated to underlie the functional organization of this complex brainstem region.

The periaqueductal gray projections to the rat spinal trigeminal, raphe magnus, gigantocellular pars alpha and paragigantocellular nuclei arise from separate neurons

Possible collateral branches of periaqueductal gray axons which distribute to the nucleus raphe magnus, nucleus reticularis paragigantocellularis, nucleus reticularis gigantocellularis pars alpha and the spinal trigeminal nucleus were analyzed with the double fluorescent retrograde tracer technique. With the exception of a small number of double-labeled neurons observed in the periaqueductal gray following injections of fluorescent dyes into the nuclei reticularis paragigantocellularis and gigantocellularis pars alpha, no double-labeled cells were found in this midbrain region following injections of tracers into various combinations of the above 4 nuclear groups. The results of this investigation indicate that these 4 brainstem nuclei are innervated predominantly by separate neuronal populations within the periaqueductal gray.

The periaqueductal gray-raphe magnus projection contains somatostatin, neurotensin and serotonin but not cholecystokinin

The retrograde transport-HRP-immunocytochemical technique was employed to ascertain if the periaqueductal gray-raphe magnus projection arises from neurons containing somatostatin, neurotensin, serotonin or cholecystokinin. Following HRP injections into the raphe magnus (NRM) double-labeled cells containing HRP reaction product and somatostatin-, neurotensin- or serotonin-like immunoreactivity were identified in the midbrain periaqueductal gray (PAG). No cholecystokinin-like immunoreactive double-labeled neurons were found in the PAG. These results indicate that the PAG-NRM pathway contains somatostatin, neurotensin and serotonin but not cholecystokinin.

Monoclonal antibody specific for carbodiimide-fixed glutamate: immunocytochemical localization in the rat CNS.

Glutamate is widely distributed in the central nervous system (CNS) and is present in greater amounts than any other putative neurotransmitter. To study its distribution in the CNS, a monoclonal antibody was raised against gamma-L-glutamyl-L-glutamic acid (gamma-Glu-Glu) conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehydeborohydride. By use of this antibody, indirect immunoperoxidase staining was observed in CNS tissue fixed with carbodiimide to form gamma-Glu-Glu from glutamate and post-fixed with glutaraldehyde or paraformaldehyde. In contrast, immunoreactivity was quite low in tissues fixed only with glutaraldehyde. Absorption controls indicated that the staining of carbodiimide-fixed tissue could be inhibited by micromolar concentrations of gamma-Glu-Glu but not by other small molecules. Using ELISA, the antibody reacted strongly with the gamma-Glu-Glu/KLH conjugate used to immunize the mouse, but not with other small molecules conjugated to KLH. The reactivity of the antibody with the gamma-Glu-Glu/KLH conjugate on ELISA was inhibited by free gamma-Glu-Glu in micromolar concentrations, but not by similar dipeptides or amino acids. Dense immunocytochemical staining was observed in cortical pyramidal cells, cerebellar granule cells, and the cochlear nuclei. Staining with this monoclonal antibody correlated well with other methods of localizing glutamate in the CNS.