Autoimmune encephalomyelitis and ocular lesions in monkeys sensitized during the neonatal period.

The neonatal rhesus monkey is susceptible to the induction of autoimmune encephalomyelitis. The disease has been produced regularly by injection of neonatal animals with guinea pig spinal cord antigen in complete Freund's adjuvant. The onset of the disease, as compared with onset in adults, is delayed and is most often heralded by intrinsic eye lesions, notably widespread retinal hemorrhages.

Primate mammary development. Effects of hypophysectomy, prolactin inhibition, and growth hormone administration.

The pituitary gland has been found to be an important factor in mammary development in primates. Hypophysectomy in 12 sexually immature monkeys caused significant inhibition of estradiol (E2)-induced mammary growth and development. A histological index of mammary development in sexually immature hypophysectomized animals was lower (0.82) than in intact E2-treated controls (3.4; P less than 0.008). Hypophysectomy also inhibited growth of the mammary gland as judged by a size index. Despite the hypophysectomy, E2 stimulated some, albeit blunted, mammary growth and development, which may have been due to incomplete hypophysectomy. Selective inhibition of prolactin by ergot drugs in intact animals did not prevent full mammary development, suggesting that there may be pituitary mammogens other than prolactin, or that very low or unmeasurable concentrations of prolactin were sufficient to synergize with E2 to cause full acinar development. The mean histological index was 3.08 in E2-treated animals and 3.16 in animals treated with E2 plus pergolide. There was also no difference in the size of the glands. We evaluated the effect of growth hormone on mammary development by treating three hypophysectomized animals with pure 22,000 mol wt human growth hormone (hGH) (Genentech, Inc., South San Francisco, CA). We found that physiological or slightly supraphysiological concentrations of hGH in animals with unmeasurable prolactin were incapable of restoring the capacity of E2 to induce full mammary growth. These findings suggest that, if growth hormone is a mammary mitogen, that physiological concentrations are insufficient to synergize with E2 to induce full mammary growth or that other forms of hGH are mammogenic. Our studies suggest that the role of the pituitary gland in mammary mitogenesis in primates is more complicated than previously thought. They also raise the possibility that heretofore unidentified pituitary substances may be mammogenic.

Acute inhibition of gonadotropin secretion by corticotropin-releasing hormone in the primate: are the adrenal glands involved?

To investigate the role of the adrenal glands in the acute inhibition of gonadotropins induced by CRH in the primate, we have compared the effects of CRH infusion on LH and FSH before and after adrenalectomy and under variable glucocorticoid backgrounds. The studies were performed in four ovariectomized rhesus monkeys. Confirming previous observations, a 5-h iv CRH (rat/human CRH, 100-150 micrograms/h), but not saline, infusion inhibited both LH and FSH secretion. Saline and CRH infusions were repeated at random intervals after adrenalectomy under each of three different glucocorticoid backgrounds, achieved by varying the glucocorticoid replacement therapy (groups 1-3). At the time of the saline or CRH tests, mean cortisol concentrations were 38.5 +/- 6.3 (+/- SE) micrograms/dl before adrenalectomy, and 21.9 +/- 1.4, 14.3 +/- 1.1, and less than 1.0 micrograms/dl in groups 1, 2, and 3 of adrenalectomized (ADX) monkeys. In response to CRH infusion, gonadotropin concentrations significantly decreased in groups 2 and 3, but not in group 1 ADX monkeys which had the highest cortisol background. By hour 5 of CRH infusion, the percentages of the preinfusion baseline area under the curves for LH were 96.8 +/- 6.2%, 44.6 +/- 3.7%, and 53.5 +/- 6.1%, for groups 1, 2, and 3; by hour 4 the values for FSH were 95.7 +/- 3.5%, 76.2 +/- 4.7%, and 74.7 +/- 4.0% for groups 1-3, respectively. The absence of a response to CRH in group 1 animals occurred even though mean cortisol concentrations were lower than those in the same monkeys before ADX. Morphine (9 mg, iv), which had previously been shown to decrease LH and FSH concentrations in ovariectomized monkeys, also significantly decreased LH and FSH concentrations in ADX monkeys of group 1, which did not respond to CRH. The maximal decline occurred by hour 3 after morphine injection, when LH and FSH areas under the curve were 51.5 +/- 11.4% and 61.0 +/- 3.2% of the preinfusion baseline. Our results clearly indicate that in the primate the adrenal glands are not required for the acute CRH inhibitory effect on LH and FSH, and consequently, the decrease in gonadotropins that follows CRH is not mediated by the resultant increase in cortisol release, but, rather, by central mechanisms. The results also show that the effectiveness of CRH in inhibiting gonadotropins in the ADX monkey is affected by the amount of glucocorticoids present at the time of the test; unexpectedly, the ADX monkey is more sensitive to this protective effect of glucocorticoids than the non-ADX animal.

Evidence that prolactin stimulates alpha-lactalbumin production in mannary tissues from premenarcheal rhesus monkeys.

PRL was found to stimulate marked increases in alpha-lactalbumin production in six of eight specimens of mammary tissue from premenarcheal rhesus monkeys, and a lesser increase was seen in a seventh. Even without added PRL, low concentrations of this milk protein (mean total alpha-lactalbumin production, 1.9 ng/dish) were released into the organ culture medium bathing these relatively immature tissues; most of the epithelial elements were ductal. Under similar conditions, significantly higher concentrations of alpha-lactalbumin (mean total production, 23.4 ng/dish) were released from tissues of sexually mature animals in which lobulo-alveolar elements were abundantly present in addition to ducts. When tissues from premenarcheal animals were exposed to ovine PRL, alpha-lactalbumin concentration in medium and tissue homogenates were increased significantly. Overall, mean total alpha-lactalbumin production rose to 12.9 (P less than 0.02) and 40.5 (P less than 0.02) ng/dish in response to 100 and 1000 ng/ml ovine PRL, respectively. In those cases in which both medium and tissue homogenates were analyzed, increases were parallel. These findings indicate that PRL has a lactogenic effect on mammary tissue from sexually immature and mature rhesus monkeys.

Duration of chorionic gonadotropin production by the placenta of the rhesus monkey.

Concentrations of macaque chorionic gonadotropin (mCG) in placenta, blood and urine of rhesus monkeys have been measured by both radioimmunoassay and bioassay throughout gestation. mCG was easily detected and quantified in these specimens for a brief period in early pregnancy, but was not detectable between the 40th day of pregnancy and term in placental extracts, serum, or 40-fold urine concentrates. The apparent absence of mCG after the 40th day of pregnancy makes these macaques a valuable model for pregnancy research, where the absence of chorionic gonadotropin is experimentally desirable. Unlike women and some higher primates, the functional status of the fetal, placental and maternal endocrine compartments of macaques can be studied in the absence of circulating chorionic gonadotropin during mid and late gestation.

Prolactin stimulation of alpha-lactalbumin in normal primate mammary gland.

An in vitro system has been developed to examine the effects of PRL on the normal primate mammary gland. alpha-Lactalbumin a milk protein, was found in breast tissue samples from 17 of 19 primates representing several Macaca and Papio species; concentrations ranged from 10-768 ng/mg protein. That none of the animals was pregnant or lactating and half were nulliparous indicates that milk protein production takes place under normal circumstances, even in breast tissue of nonlactating animals. Studies of the effect of PRL on alpha-lactalbumin production in these tissues in organ culture revealed that PRL maintained existing or stimulated new production of alpha-lactalbumin for periods of up to 9 days. Measurement of alpha-lactalbumin in medium bathing mammary tissue from three animals revealed that mean alpha-lactalbumin production during days 7-9 when PRL was added (100 and 1000 ng/ml) was 11 and 59 times greater, respectively, than control. Simultaneous measurement of tissue concentrations of alpha-lactalbumin revealed that those tissues maintained with PRL (1000 ng/ml) had a mean concentration of alpha-lactalbumin that was 61 times that of controls without PRL. PRL consistently maintained or increased alpha-lactalbumin production in tissues from all 22 primates tested. Even in those premenarchal animals in whose mammary tissue alpha-lactalbumin was undetectable initially, PRL stimulated alpha-lactalbumin production in a dose-related fashion. In contrast, when PRL was absent from medium, alpha-lactalbumin concentrations decreased at 9 days to less than 20% of the initial 3-day value in all cases. These studies provide evidence that mammary tissue from normal nonlactating, nonpregnant primates produces milk proteins and that when tissues are exposed to PRL in culture, production of alpha-lactalbumin is stimulated.

Alcohol-induced brain changes in dogs.

We studied the neuropathologic effects of chronic alcohol ingestion on the brains of healthy, well-nourished, male mongrel dogs. Five experimental dogs were provided 36% of their calories as ethyl alcohol for 1 year. Following killing, their brains were weighed, photographed, sectioned, and processed for computerized morphometric determinations of ventricular size, cortical thickness, and neocortical neuron and glial cell populations. Compared with a similarly handled control group, the alcoholic dog brains showed lateral ventricular enlargement, cortical thinning in the temporal lobe only, and fewer glial cells in the temporal and frontal cortices. There were no statistically significant differences between the alcoholic and control groups in brain weight, frontal or parietal cortical thickness, or neocortical neuron populations. These results imply a disproportionate vulnerability of white matter to the damaging effects of alcohol with consequent lateral ventricular enlargement, and some regional variation in neocortical susceptibility to alcohol-induced cortical thinning and glial cell loss. In general, such changes are consistent with those described in neuroradiologic imaging studies of human alcoholics.

Differential effects of antiepileptic drugs and beta-carbolines on seizures induced by excitatory amino acids.

Agonists acting at subtypes of glutamate receptors, N-methyl-D-aspartate, kainate and quisqualate, induce convulsions in rodents. Clonic seizures induced in mice by intracerebral administration of N-methyl-D-aspartate, kainate or quisqualate were used to study the anti- and proconvulsant potential of antiepileptic drugs and beta-carbolines. Systemic administration showed that the benzodiazepines clonazepam and midazolam blocked convulsions induced by kainate and had no effect on seizures triggered by N-methyl-D-aspartate and quisqualate. In contrast, diazepam blocked convulsions induced by either excitatory amino acid, as did valproate. The benzodiazepine receptor agonist beta-carboline ZK 93423 blocked convulsions induced by kainate but had no effect on seizures induced by N-methyl-D-aspartate or quisqualate. The antagonist beta-carboline ZK 93426 did not affect convulsions induced by excitatory amino acids, while the inverse agonists FG 7142 and ethyl-beta-carboline-3-carboxylate increased the sensitivity of mice to kainate. Phenobarbital and 2-chloroadenosine protected mice against seizures induced by quisqualate and kainate, while baclofen was active against convulsions produced by kainate. MK-801 selectively blocked convulsions induced by N-methyl-D-aspartate, and enhanced the susceptibility of mice to seizures triggered by kainate and quisqualate. Ethosuximide increased the susceptibility of mice to N-methyl-D-aspartate and had little or no effect on other types of seizures. Diphenylhydantoin enhanced the convulsant potential of quisqualate. Trimethadione and carbamazepine did not affect convulsions induced by N-methyl-D-aspartate, kainate or quisqualate. Intracerebral administration of midazolam protected mice against seizures induced by kainate. Ethosuximide increased the susceptibility of mice to N-methyl-D-aspartate, while diphenylhydantoin to quisqualate convulsions.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical and ultrastructural changes in tapetum of beagles with a hereditary abnormality.

We have defined the defect in the tapetum of beagles with an inherited tapetal abnormality to be the absence of both zinc and cysteine from the tapetal rods. This was demonstrated by direct measurement of zinc and cysteine and by histochemical localization of zinc. The latter method also indicated an interesting substructure in tapetal rods from normal beagles, in which the zinc is apparently distributed in two concentric rings. The chemical changes involved in different methods of fixing this unique tissue have been examined and correlated with the ultrastructure.

Physicochemical stability of intravenous lipid emulsions as all-in-one admixtures intended for the very young.

BACKGROUND & AIMS: Intravenous lipid emulsions (IVLEs) are unstable when growth of lipid droplets into large fat globules is detected by appropriate particle sizing techniques. Specifically, instability is evident when the volume-weighted percent fat (PFAT)>5 microm exceeds 0.4% of the total lipids present. This represents an approximate 10-fold increase in the population normally present in the large-diameter tail of stable lipid emulsions. The composition of the oil phase of an IVLE, however, has been shown to exhibit different stability characteristics. We investigated the stability of various IVLEs containing physical mixtures of medium-(MCT) and/or long-chain triglycerides (LCT) in three different all-in-one (AIO) admixtures intended for neonatal and infant patients. METHODS: The 20% (w/v) IVLEs used in this study were composed of the following oils (by weight): 1). 1:1-soybean/safflower (SS); 2). 1:1-MCT:soybean (MS); and 3). 5:4:1-MCT:soybean:fish (MSF). Stability was assessed by light obscuration or light extinction to count large fat globules, and by aided (microscopic) and unaided (naked eye) visual assessments for up to 48 h at room temperature. RESULTS: The stability of SS-based admixtures significantly and rapidly deteriorated in one of the three AIO compositions studied, whereas the AIOs made from MS or MSF were stable for all formulations. CONCLUSION: The results suggest that AIOs made from MCT/LCT-containing IVLEs are more stable than those made from pure LCTs.

Physicochemical stability assessments of lipid emulsions of varying oil composition.

BACKGROUND AND AIMS: Intravenous lipid emulsions have been shown to be unstable when the percent fat >5 microm (PFAT >5 microm) exceeds 0.4% by weight of the total fat present. We investigated the physicochemical stability of a standard low amino acid and carbohydrate mixture containing electrolytes when combined with four different commercial intravenous lipid emulsions of varying oil composition. METHODS: The 20% (w/v) lipid emulsions studied were composed of the following oils (by weight): 1) 1 : 1 soybean/safflower (SS); 2) 100% soybean (S); 3) 1 : 1 soybean/MCT (SM) and 4) 4 : 1 olive/soybean (OS). Physicochemical stability was assessed by light obscuration or extinction using a single-particle optical sensing technique to detect growth of fat globules in the large diameter tail (>1 microm) of the droplet size distribution and by visual analyses for evidence of phase separation. RESULTS: The physicochemical stability of SS and S-based all-in-one mixtures significantly deteriorated over time when compared to the mixtures made from SM and OS. In addition, of the four mixtures studied that contained SS (n=2) and S (n=2), only one of each bag studied showed visually obvious destabilization by the presence of free oil from phase separation, despite highly abnormal changes in the globule size distribution of all four preparations. CONCLUSION: The results suggest that all-in-one mixtures composed of either soybean oil alone or in combination with safflower oil are less stable than those mixed with either MCT or olive oil which also contain sodium oleate that can act as co-emulsifying agent.

Physicochemical assessments of parenteral lipid emulsions: light obscuration versus laser diffraction.

The United States Pharmacopeia (USP) has proposed a new Chapter <729> entitled 'Globule Size Distribution in Intravenous Emulsions' that is intended to identify methods for analyzing the stability of lipid emulsions. We studied the differences between particle-sizing instruments when analyzing the physicochemical stability of a parenteral nutrition mixture compounded with intravenous lipid emulsion, known as an all-in-one mixture. As the growth of lipid droplets, i.e. coalescence, signals an irreversible change in emulsion stability, we focused our investigation on the large diameter tail (>5 microm) of the globule size distribution. Of the four proposed methods, droplet size was studied over a range of mixture stabilities using a low osmolality parenteral nutrition formula employing both light scattering and light obscuration techniques. In addition, the same mixtures were also freshly prepared, and then spiked with a known amount of 5 microm latex spheres. The response obtained from the light obscuration technique was linear and detected both unstable and latex-spiked mixtures in every case for droplets or particles >5 microm. The results of the laser diffraction method were non-linear and overestimated, was less sensitive or missed entirely, globules or particles in the large diameter tail of the dispersion. The results demonstrate that light obscuration is superior to laser diffraction in identifying unstable intravenous fat emulsions.