GABA(B) receptor-mediated selective peripheral analgesia by the non-proteinogenic amino acid, isovaline.

Peripherally restricted analgesics are desirable to avoid central nervous system (CNS) side effects of opioids. Nonsteroidal anti-inflammatory drugs produce peripheral analgesia but have significant toxicity. GABA(B) receptors represent peripheral targets for analgesia but selective GABA(B) agonists like baclofen cross the blood-brain barrier. Recently, we found that the CNS-impermeant amino acid, isovaline, produces analgesia without apparent CNS effects. On observing that isovaline has GABA(B) activity in brain slices, we examined the hypothesis that isovaline produces peripheral analgesia mediated by GABA(B) receptors. We compared the peripheral analgesic and CNS effect profiles of isovaline, baclofen, and GABA (a CNS-impermeant, unselective GABA(B) agonist). All three amino acids attenuated allodynia induced by prostaglandin E2 injection into the mouse hindpaw and tested with von Frey filaments. The antiallodynic actions of isovaline, baclofen, and GABA were blocked by the GABA(B) antagonist, CGP52432, and potentiated by the GABA(B) modulator, CGP7930. We measured Behavioural Hyperactivity Scores and temperature change as indicators of GABAergic action in the CNS. ED(95) doses of isovaline and GABA produced no CNS effects while baclofen produced substantial sedation and hypothermia. In a mouse model of osteoarthritis, isovaline restored performance during forced exercise to baseline values. Immunohistochemical staining of cutaneous layers of the analgesic test site demonstrated co-localization of GABA(B1) and GABA(B2) receptor subunits on fine nerve endings and keratinocytes. Isovaline represents a new class of peripherally restricted analgesics without CNS effects, mediated by cutaneous GABA(B) receptors.

Weight change during lactation does not alter the concentrations of chlorinated organic contaminants in breast milk of women with low exposure.

The purpose of this longitudinal study was to evaluate whether a loss of body fat during lactation between 4 and 20 wk postpartum increases the concentration of environmental contaminants in breast milk. We examined this relationship in two different cohorts of exclusively breastfeeding women: (1) California women with low exposure to contaminants (n = 10 whose weight was stable [mean change, 0.0 +/- 0.2 kg] and n = 11 who lost weight [mean loss, -4.1 +/- 0.4 kg]) and (2) Hispanic women (n = 30) who had recently immigrated to North Carolina, primarily from Mexico, and were presumed to have a higher prior exposure to contaminants. Breast milk samples were analyzed for hexachlorobenzene (HCB) and DDT and its related products. There was no significant relationship between change in body composition and change in milk contaminant concentrations in either California or North Carolina women. Concentrations of HCB were similar between the California and North Carolina women, but those of DDT and related products were higher in the North Carolina subjects. Results suggest that moderate weight loss in lactating women with low exposure to environmental contaminants does not increase contaminant concentration in breast milk.

Age-related alterations in the morphology of femoral artery vasa vasorum in the rat.

The objective of this study was to explore any age-related morphological changes in the vasa vasorum of the rat femoral artery. Vascular corrosion casts were prepared from 2, 12 and 24-month-old rats. Examination of the casts with the scanning electron microscope revealed dramatic differences in the appearance of the vessels of young and aged rats. The vasa vasorum of 2-month-old rats consisted of a dense network of capillaries. These vessels were dramatically reduced in number by 12 months, and even fewer capillaries were present at 24 months. This reduction in capillary density is consistent with the observed age-related decreases in oxygen tension and may explain why the aged are more prone to atherosclerosis.

Vascular endothelial growth factor (rhVEGF165) stimulates direct angiogenesis in the rabbit cornea.

The object of this study was to test vascular endothelial growth factor (VEGF) for angiogenic activity in the rabbit corneal assay. VEGF doses ranging from 20 ng to 1000 ng were incorporated into a slow release polymer and implanted into the avascular rabbit cornea. Capillary formation in the cornea was visually analyzed on a daily basis and examined with histology, transmission electron microscopy and scanning electron microscopy of vascular corrosion casts on days 2 and 7 post-implantation. VEGF implants (200ng to 1000ng) consistently stimulated angiogenesis. This neovascularization occurred in the absence of inflammation. We conclude that VEGF acts directly on endothelial cells, initiating and mediating the formation of capillaries.

Platelet derived wound healing factors (PDWHF) accelerate and augment wound healing angiogenesis in the rat.

The object of this study was to determine the effect of PDWHF on wound healing angiogenesis. Sponges saturated with PDWHF were implanted in rats. Six hours to 14 days post-implantation, vascular corrosion casts were prepared and examined by SEM. Leukocyte margination and angiogenesis was accelerated by 24-48 hours. Vessel number was also greater than in the controls. Another series of PDWHF treated sponges were implanted. Fourteen days later the vasculature was perfused with Evans blue. Sponges were homogenized, and absorbance of the supernatants was determined. Absorbances of PDWHF supernatants was 2X greater than controls. PDWHF enhanced wound healing angiogenesis in the rat.

Transforming growth factor beta (TGF-B) stimulation of angiogenesis: an electron microscopic study.

The object of this study was to examine the initiation and pattern of corneal angiogenesis stimulated by transforming growth factor beta (TGF-B) using scanning electron microscopy (SEM) of vascular corrosion casts and transmission electron microscopy (TEM). Fifty nanograms of TGF-B was combined with the slow release polymer Hydron and implanted in the rabbit cornea as an assay for angiogenic activity. Two and 7 days after implantation, the corneas were removed for TEM. At the same time intervals the limbal vasculature was filled with Mercox, an acrylic monomer. After the Mercox hardened, the tissue was digested with alternating immersions in 40% KOH and distilled water. The resulting casts were air-dried, sputter coated with gold and viewed with the scanning electron microscope. TEM revealed that 2 days after implantation, TGF-B elicited an influx of a vast number of inflammatory cells, which at two days were predominantly neutrophils. By 7 days the inflammation had subsided and typical capillaries were seen between the collagenous laminae of the normally avascular corneal stroma. SEM of vascular corrosion casts showed evidence of margination/diapedesis of leukocytes from the limbal venules 2 days post-implantation. In addition, the casts from the 7 day time point showed that new vessels arose solely from limbal venules.

Macrophage colony-stimulating factor induces indirect angiogenesis in vivo.

The cytokine macrophage colony-stimulating factor was implanted in the rabbit cornea over a wide dose range (1 ng to 100 microg) to assay its angiogenic activity in vivo. Neovascularization occurred in a dose-dependent manner, and maximum angiogenesis occurred only with 100 microg. Histologic analysis revealed that the corneas were free of inflammation at the lower doses, but had slight inflammation at 50 and 100 microg. Nonspecific esterase staining of frozen sections and transmission electron microscopy revealed that the inflammatory cells were predominantly macrophages, with very few neutrophils present. This association of capillary formation with inflammation suggests an indirect mechanism of angiogenesis. The lack of neutrophils within the inflammatory cell infiltrate demonstrates that indirect angiogenesis can proceed without the local presence of neutrophils. This distinguishes macrophage colony-stimulating factor from other indirect-acting angiogenesis factors that have been identified to date.

Inhibition by methylprednisolone acetate suggests an indirect mechanism for TGF-B induced angiogenesis.

Angiogenesis induced by transforming growth factor beta (TGFB) implanted in the rabbit cornea is accompanied by an influx of inflammatory cells. To determine if the inflammatory cells are the mediators of the neovascularization, they were depleted by local administration of methylprednisolone acetate (MPA). Subconjunctival injections of 16 mg of MPA immediately following implantation of 50 ng of TGFB in the cornea prevented the inflammation and subsequent formation of capillaries. If the injections of MPA were delayed by 48 hr and the inflammatory cells were allowed to enter the cornea, angiogenesis occurred, demonstrating that MPA had no adverse effects on the ability of endothelial cells to form capillaries. These results confirm the hypothesis that TGFB induces angiogenesis indirectly by recruiting inflammatory cells capable of stimulating direct angiogenesis.

Initiation and pattern of angiogenesis in wound healing in the rat.

The object of this study was to examine the initiation and pattern of capillary growth associated with wound healing. Collagen sponges were implanted subcutaneously in the hind limbs of adult male rats to stimulate the formation of granulation tissue. Blood vessels of the hind limbs of euthanized rats were perfused with Mercox (an acrylic monomer) via the abdominal aorta at selected periods of time following sponge implantation. When the perfusate was completely cured, the sponge and parajacent tissues were excised and subsequently macerated by alternating immersion in 40% KOH and distilled water. Cast replicas of the vascular lumina were coated with gold and imaged by scanning electron microscopy. At 6 hr, punctate depressions at the periphery of the replicas of vein and venule lumina were noted. The depressions represented sites of leukocyte margination. By 24 hr, the depressions increased numerically, indicating a great increase in the sites of leukocyte margination. The number of these depressions decreased by 48 hr. Concomitantly, the depressions representing endothelial cell nuclei became more pronounced, indicating nuclear hypertrophy of these cells. In addition, capillary bud formation was initiated. At 72 hr, capillary buds were quite apparent and arose solely from venules. Between 7 and 14 days, replicas of capillary lumina were longer and formed an elaborate network, presumably by end-to-end, side-to-side, and end-to-side anastomoses. The network was formed circumferential to the sponge and then capillary sprouts entered the sponge's interstitial spaces.