Experimental protein malnutrition in primates--cytochemistry of the nervous system

Experimental protein malnutrition was induced in groups of young juvenile squirrel monkeys by feeding them ad libitum diets very low in protein content, whereas a diet containing 25% protein content was fed to the control animals. Detailed cytochemical studies have clearly shown the sensitivity of the nervous system to dietary abuse. The motor neurons of the spinal cord and the Purkinje cells of the cerebellum are very sensitive to protein deficiency, and the number of oligodendroglia cells increases sharply. Gallocyanin stained preparations from the malnourished animals show significant decrease in the amount of RNA in the Purkinje cells of cerebellum and the anterior horn cells of the spinal cord. The larger neurons show prominent chromatolysis with concomitant increase in the number of oligodendrocytes surrounding such nerve cells. The different layers of the cerebellar cortex of the malnourished animals, as well as some of the neurons of the spinal cord, showed decreased activity of succinate dehydrogenase and increased levels of thiamine pyrophosphatase and lactate dehydrogenase. Although ATPase activity appeared to remain unchanged quantitatively, it showed profound disturbance in its in situ localization, especially in the mitochondrial ATPase located in the cytoplasm. The enzyme activity in the nuclear membrane appeared unchanged. These studies emphasize the importance of studying in situ changes in the anatomically heterogeneous nervous tissue, rather than of working only with biochemical methods using homogenized material.     

Nucleolar activity in the primate dorsal root ganglion cells associated with dietary protein malnutrition.

The nucleolar changes in the dorsal root ganglion cells of the squirrel monkey, Saimiri sciureus, have been investigated by the use of histological and histochemical methods in the healthy neonates (born to mothers maintained on 25% protein diet during gestation) and young adults, and compared with those observed in the protein-malnourished neonates (born to mothers maintained on a 8% protein diet during gestation). Also studied were young adults maintained on a high protein diet (25% protein) as well as on a low protein diet (2% protein content) for a period of 15 weeks. Whereas in the healthy animals (neonates and young adults) only a few cells show nucleolar budding and finally their extrusion into the cytoplasm, the protein-malnourished animals show a significantly enhanced nucleolar activity in the form of increased size (3-4 times the original) and active budding either from one focal point or at several places on the body of the nucleolus. In the neurons which show chromatolytic changes, these extrusions migrate to the nuclear membrane, wherefrom they pass on to the cytoplasm through an evagination of the nuclear envelope. Such a migration appears to occur by a focal dissolution of the nuclear membrane. It appears that, under conditions of severe malnutrition and advanced chromatolytic changes in the neuronal cytoplasm, the nucleolar material provides a compensatory mechanism for the increased cytoplasmic catabolism and loss of ribonucleoprotein material in order to facilitate additional protein synthesis for cell survival.     

Experimental protein malnutrition in squirrel monkeys: cytochemical interaction of nucleolus and cytoplasm in the anterior horn cells of the spinal cord of Saimiri sciureus.

Nuecleolo-cytoplasmic relationships have been studied using histochemical techniques in the spinal cord of healthy neonates compared with those born to mothers malnourished through most of the gestation period, and healthy and severely protein-malnourished young adult squirrel monkeys. Formaldehyde-fixed as well as fresh frozen tissues were used for histological study and for histochemical techniques to demonstrate lipids, carbohydrates, nucleic acids and enzymes of various metabolic cycles. Changes in the neuronal cytoplasm of the low protein animals appear to be related to impaired protein metabolism and energy transport. The nucleolus reacts to cytoplasmic changes by enlarging its size and by active synthesis of secretion of nucleolar material, which is then passed into the cytoplasm through an evagination of the nuclear envelope. The greater the degree of chromatolysis in the cytoplasm, the more active the nucleolus appears to be in terms of its enlarged size and transfer of secretion products into the cytoplasm. It is believed that the perinuclear accumulation of secretion products in the neuronal cytoplasm of the protein-deficient animals has its origin in the nucleolus. Such an accumulation in the severely malnourished animals appears to be a compensatory mechanism for the increased cytoplasmic catabolism and loss of ribonucleoprotein material in order to facilitate additional protein synthesis for cell survival.     

In vitro incorporation of (U-C14)-glucose and (1-C14)-sodium acetate in peripheral nerves of malnourished young rhesus monkeys

The effect of protein calorie malnutrition (PCM) on synthesis of lipids in peripheral nerves was studied by in vitro incorporation of (U-C14)-glucose and (1-C14)-sodium acetate. Ulnar and tibial nerves obtained from five young rhesus monkeys with PCM, five rehabilitated monkeys, and five control monkeys were incubated for 2 h with the radioactive precursors. Uptake of both radioactive precursors in whole peripheral nerves as well as myelin marker lipids was significantly decreased in animals with PCM. However, uptake returned to normal in rehabilitated monkeys.     

Experimental protein malnutrition in squirrel monkeys: reaction of the nissl substance in the motor neurons of the spinal cord

Summary Experimental protein malnutrition was induced in juvenile squirrel monkeys by feeding them synthetic diets which contained 2% protein for a period of 15 weeks. Along with their controls who were fed 25% protein food and laboratory monkey chow, groups of monkeys were sacrificed at nine, 11, 13, and 15 weeks of feeding schedule. The spinal cords were stained with chromalum-gallocyanin, PAS, Trichrome Masson's method and Alcian Blue. The neurons and the surrounding glial cells showed significant cytoohemical changes in different groups of the malnourished animals. The motor neurons of the spinal cord showed a varying but dramatic reaction to protein deficiency, which reflects a profound physiological distortion. These observations have been discussed in relation to probable functional derangement under the influence of protein deficiency.     

Regional distribution of homocarnosine and other ninhydrin-positive substances in brains of malnourished monkeys

—Eight male monkeys (Macaca nemestrina) aged 6–9 months were divided into two groups and fed either an adequate protein diet (20% casein) or a protein deficient diet (2% casein). After 3- 5 months of receiving the low protein diet, the malnourished monkeys showed extensive fatty metamorphosis of the liver cells, distorted patterns of plasma and hepatic free amino acid pools, and other features consistent with the diagnosis of protein-calorie malnutrition. Examination of the cerebrum, cerebellum and brain stem in the malnourished animals revealed profound accumulation of 3-methylhistidine, histidine and homocarnosine in all three regions. For histidine, the cerebral, cerebellar and brain stem levels in the protein deficient animals increased by 145, 104 and 101 per cent over levels observed in corresponding regions of the brain in well-fed monkeys. Similarly, there were significant elevations in homocarnosine contents of the cerebrum (+ 99 per cent), cerebellum (+ 140 per cent) and brain stem (+ 146 per cent) in comparison to levels in control animals. In contrast, the levels of valine, serine and aspartic acid were markedly reduced in all three brain areas in the malnourished animals. Protein-calorie deficiency also produced reductions in the brain levels of taurine, glutamic acid, isoleucine, leucine and threonine which varied in magnitude in the three major regions of the brain examined. These biochemical alterations which may in part underlie some of the psychomotor changes often observed in protein-calorie malnutrition, were discussed not only in relation to the role of amino acids as precursors for the synthesis of neuroregulatory substances but also with due regard to the possibility that some of these ninhydrin-positive substances such as GABA, homocarnosine, glycine and the dicarboxylic amino acids may possess neuroexcitatory or inhibitory properties in various parts of the central nervous system.     

Changes in the content and concentration of cytoplasmic and nuclear proteins in nerve and glial cells of hypothalamic nuclei at different times of water deprivation

The data obtained show that searching activity of animals increases at an early stage (24 hrs) of water deprivation (WD) and is accompanied by an increase in the content of cytoplasmic proteins. WD up to 3 days produces further rise of motor searching activity with simultaneous increase of protein content not in the cytoplasm alone but in the nuclei of neurons and glial cells as well. During long-term periods of WD (5 days), motor activity considerably decreases, the animals showing signs of depression. The content and concentration of proteins significantly decrease in all studied components of neurons and glia of the hypothalamic nuclei under study. Existence of correlation between protein content (cytoplasmic and nuclear) and behavior of animals is discussed.     

Nuclear and cytoplasmic RNA in visual cortex neurons of adult rats following visual deprivation and photic stimulation

It has been shown by two-wavelength cytospectrophotometry of gallocyanin-chrome alum-stained sections that visual deprivation in adult rats kept in a complete darkness for 30 days resulted in an accumulation of cytoplasmic RNA by layer V neurons of the visual cerebral cortex and by the cells of the perineuronal neuroglia of this layer. The nuclear RNA content remained unchanged. Stimulation of intact rats with a flickering or constant light induced an increase in the cytoplasmic RNA in these neurons rather than in the nuclear RNA as well as in RNA in their glial satellite cells. Similar light stimulation of the deprived animals gave rise to a complete return of the neuronal RNA to normal with only a slight decrease in the deprivation-induced RNA accumulation by the neuroglial cells. Neither visual deprivation nor light stimulation affected the RNA content in the neurons and neuroglia of layer V of the motor cerebral cortex. Compartmentation of RNA metabolism within the neuronal-neuroglial unit is discussed.     

Experimental protein malnutrition in primates: Cytochemical studies on the cerebellum of the squirrel monkey,Saimiri Sciureus

Synopsis The cerebellum of healthy and malnourished squirrel monkeys was studied histopathologically and cytochemically for a number of important enzymes such as phosphatases (acid and alkaline phosphatase, ATPase, thiamine pyrophosphatase), esterases (simple esterase and acetylcholinesterase), dehydrogenases (succinate, malate and isocitrate dehydrogenase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase), monoamine oxidase and phosphorylase. The Purkinje cells, stellate and basket cells were found to be more sensitive to protein malnutrition compared to the other types of cells in the cerebellum. An increase in the number of dark cells with large amounts of ribonucleoprotein complex in the Purkinje cell layer of the extremely malnourished animals sacrificed after 15 weeks on a low protein diet may be significant and may reflect either an abnormal metabolic process or an interruption in the axonal transport of RNA complex. This may also be directly related to a significant reduction in the level of oxidative enzymes, especially those of the tricarboxylic acid cycle, these being the main source of energy stored in ATP. At the same time the level of lysosomal enzymes, which are responsible for the catalysis of the different degradation reactions, is greatly increased and indicates cellular catabolism. The present investigations point to the probability that the neurons adapt to the changed environment by beginning to utilize structural proteins for their basic metabolism.     

Measures of body size and growth in rhesus and squirrel monkeys subjected to long-term dietary restriction

Although many studies have reported the robust effects of dietary restriction (DR) in retarding numerous aging processes in rodents, little is known about the outcomes of reducing caloric intake of a nutritious diet on aging in primates. Most primate studies have concerned the effects of malnutrition. We hypothesized that DR influences aging processes in primate species as it does in rodents. In the present study, 24 male rhesus (Macaca mulatta) monkeys (ages 0.6–5 years) and 25 male squirrel (Saimiri sp.) monkeys (ages 0.3–10 years) were provided diets formulated differently for each species but both fortified with vitamins and minerals (40% above recommended levels) as controls (approximating ad libitum levels) or experimentals (about 30% below the level of diet provided controls of comparable age and body weight). The results reported here concern the hypothesis that DR imposed during various developmental stages in these two primate species would affect morphometric parameters obtained at different occasions during the first 5 years of the study. Groups of older monkeys (rhesus: 18–25 years, n = 3; squirrel: 10–15 years, n = 4) were also included as controls for comparative purposes. Among groups of rhesus monkeys begun on DR prior to 6 years of age, growth in body weight and crown-rump length was reduced about 10–20% beginning after 1 year on the diet, with estimated food intake being reduced about 30–35% over this period. Measures of skin-fold thickness and various body circumference measures were also reduced in experimental groups of rhesus monkeys. In contrast, the DR regimen involving a different diet produced little impact on comparable measures in squirrel monkeys, with the estimated food intake being reduced only about 20–25% over this period. However evidence of divergence in some morphometric parameters in squirrel monkeys was beginning to emerge in young groups (<5 years(after 3 yers on the diet. © 1995 Wiley-Liss, Inc.     

Experimental protein malnutrition as a causative factor in the histological and histochemical disruption of the ependymal cells of the third ventricla and cervical central canal of squirrel monkeys.

The histologic disturbances of the cuboidal and columnar ependymal cells of the neonates under the extrinsic influence of maternal protein deprivation during most of the gestation period have been demonstrated in the central canal of the cervical spinal cord and III ventricle of the squirrel monkey brain. The control animals whose mothers were maintained on high protein diets showed an unbroken ependymal layer with an intact glial fiber layer and the subependymal cell plate. Taking birth-weight as an indication of the degree of malnutrition, maximum disruption of the ependymal layer was observed in those animals born around 80 g birth-weight. In the latter, most of the ependymal cells lost their characteristic arrangement and columnar shapes. The ependymal cells of the manourished animals show a marked reduction of oxidative enzyme content and relatively active glycogen metabolism as compared to the healthy controls. The significance of these changes has been discussed in the light of parallels in the embryological development and, to some extent, functions between the orinary neurons, ependymal cells of the choroid plexus, other ependymal cells and the glial cells. In this context, the changes observed in the present studies under the effect of protein malnutrition must be taken as part of overall changes experienced by various kinds of cells comprising the nervous system.     

Lipid composition of the peripheral nerves in malnutrition: an experimental study in young rhesus monkeys

An experimental model of protein calorie malnutrition (PCM) was created in young rhesus monkeys. Ulnar and tibial nerves from six monkeys with PCM, six rehabilitated and 12 control monkeys were studied for lipid composition and activity of myelin marker enzyme. Total lipids, myelin marker lipids and activity of myelin marker enzyme, 2', 3'-cyclic nucleotide 3' - phosphohydrolase were decreased, while esterified cholesterol and free fatty acids were increased in PCM as compared to controls. There was partial recovery in myelin marker lipids and complete restoration of other lipids on nutritional rehabilitation.     

Vitamin C deficiency in captive nonhuman primates fed commercial primate diet

Scurvy was diagnosed in 19 rhesus monkeys (Macaca mulatta) and four squirrel monkeys (Saimiri sciureus) from a colony of nonhuman primates maintained on a commercial diet. Signs of weakness, reluctance to move, gingival hemorrhage, bruising, proximal and distal metaphyseal fractures, weight loss and anemia appeared in juvenile and young adult rhesus monkeys over a 2 week period. Clinical signs subsided after 5 days of vitamin C therapy. At the same time, cephalohematomas and weakness developed in squirrel monkeys, which failed to respond to treatment. These cases were associated with manufacturer's admitted error in preparation of the commercially prepared monkey diet.     

Experimental protein malnutrition during gestation and breeding performance of squirrel monkeys,Saimiri sciureus

The report summarizes our four year experience with the breeding of squirrel monkeys, a New World species used widely in experimental research. Breeding squirrel monkeys in captivity has hitherto been considered capricious, with success in most instances not exceeding 50%. By a combination of hand picking the animals, housing in a harem-type situation with one male to several females, and sound nutrition, a success rate of 85–91% live births to number of pregnancies has been achieved. The rate of stillbirths and abortions in the group maintained on a well-balanced diet did not exceed 10% of the total pregnancies. This compares favorably with any successful breeding program with Old World monkeys, whose reproductive physiology is relatively well understood. It is clear from our studies that adequate quantities of proteins are essential for optimum reproduction. Among the pregnant animals maintained on a diet containing about 50% of their normal requirements of protein (8% calories from a protein source), the incidence of abortions increases significantly. In this group, the rate of abortions to total pregnancies ranges from 40.5 to 61.5%. The females maintained on a protein deficient diet during pregnancy are so protein hungry that an extruded fetus is a welcome food and no time is lost in devouring all of the placenta and the fetus. This type of cannibalism has never been observed among the mothers maintained on a high protein regimen.     

Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet

The in vivo effects of protein malnutrition and protein rehabilitation on lactase phlorizin hydrolase (LPH) synthesis were examined. Five-day-old pigs were fed isocaloric diets containing 10% (deficient, n = 12) or 24% (sufficient, n = 12) protein. After 4 wk, one-half of the animals in each dietary group were infused intravenously with [(13)C(1)]leucine for 6 h, and the jejunum was analyzed for enzyme activity, mRNA abundance, and LPH polypeptide isotopic enrichment. The remaining animals were fed the protein-sufficient diet for 1 wk, and the jejunum was analyzed. Jejunal mass and lactase enzyme activity per jejunum were significantly lower in protein-deficient vs. control animals but returned to normal with rehabilitation. Protein malnutrition did not affect LPH mRNA abundance relative to elongation factor-1alpha, but rehabilitation resulted in a significant increase in LPH mRNA relative abundance. Protein malnutrition significantly lowered the LPH fractional synthesis rate (FSR; %/day), whereas the FSR of LPH in rehabilitated and control animals was similar. These results suggest that protein malnutrition decreases LPH synthesis by altering posttranslational events, whereas the jejunum responds to rehabilitation by increasing LPH mRNA relative abundance, suggesting pretranslational regulation.     

Differential effects of protein malnutrition and ascorbic acid deficiency on histidine metabolism in the brains of infant nonhuman primates

Abstract: Male infant nonhuman primates (M. nemes-trina) born in captivity were used in the study. They were divided into three groups. The first group of three animals was fed a 20% casein diet and the second group of six monkeys received a 2.0% casein diet. The third group of four monkeys received a 20% casein diet totally devoid of ascorbic acid for 3.5 weeks before the diet was supplemented with ascorbic acid (20 mg/kg diet). All the diets were given to the animals in two daily rations of 100 g/animal. The monkeys fed a 2% casein diet failed to grow, and after about 3.5 months showed variable degrees of edema, hypoalbuminemia, evidence of psychomotor disturbance, depressed plasma levels of many essential amino acids, and other features consistent with the diagnosis of protein-energy malnutrition. Examination of the brains revealed significant alterations in the levels of histidine (+ 172%) and homocarnosine (+ 146%) in comparison with the control well-fed monkeys. Associated with the increase in brain histidine was a marked elevation of brain histamine level. Protein deficiency also led to poor brain retention of ascorbic acid but not to the same degree observed in the ascorbic acid-deficient animals. The latter group of animals, after receiving their diet for about 8 months, demonstrated a modest elevation in the plasma levels of most amino acids in comparison with controls. Ascorbic acid deficiency elicited a significant reduction (p < 0.01) in brain level of histidine, with hardly any change in homocarnosine level. In addition, vitamin C deficiency produced elevation of brain histamine level comparable to findings in the protein-energy-deficient monkeys. The results suggested that protein deficiency raised brain histamine level mainly through increased availability of the precursor amino acid histidine, while defective degradation might account for the increased brain level of this amine in ascorbic acid-deficient monkeys. Histamine has been proposed to have a predominantly depressant action on relevant neurons, and has also been shown to participate with other neuro-transmitters in influencing the function of the pituitary gland by regulating release of the hypothalamic hormones into the portal vessels. The relevance of the findings of marked increases in brain histamine in experimental protein and ascorbic acid deficiencies to the behavioral and extensive endocrinological alterations seen in human malnutrition deserves some intensive investigation.     

Elevation of brain histamine content in protein-deficient rats.

—Male rats of the Sprague-Dawley strain (80–250 g body wt) were fed either an adequate protein diet (18% lactalbumin) or a protein-deficient diet (0.5% lactalbumin). After 5–8 weeks of receiving the low protein diet, some of the malnourished rats were rehabilitated with an adequate protein diet. The malnourished rats exhibited significant elevations in brain levels of histidine (+415%) and homocarnosine (+100%) in comparison to findings in the control animals of similar age. Associated with the elevated brain levels of histidine in malnutrition was a prominent increase in brain content of histamine (+ 150-+ 238%). The mean brain histamine levels (ng/g) in the control rats varied from 45.96 to 56.15 in several experiments. In the protein-deficient rats, values ranged from 115 to 190. Refeeding the malnourished rats with adequate protein diet elicited reversal of histidine and histamine levels to near normal values within 1 week. The increased brain content of histamine in malnutrition was attributed to enhanced rate of production resulting from increased availability of the precursor amino acid, a conclusion consistent with elevation also of the brain content of homocarnosine (γ-aminobutyryl-l -histidine) which is another major route of disposal of histidine in the brain. The relevance of these neurochemical alterations to the behavioural changes often associated with protein malnutrition, deserves some intensive examination.     

Comparison of rectal and tympanic core body temperature measurement in adult Guyanese squirrel monkeys (Saimiri sciureus sciureus)

Background Measuring core body temperature in a manner that is safe for animals and veterinary personnel is an important part of a physical examination. For nonhuman primates, this can involve increased restraint, additional stress, as well as the use of anesthetics and their deleterious effects on body temperature measurements. The purpose of this study was to compare two non‐invasive methods of infrared tympanic thermometry to standard rectal thermometry in adult squirrel monkeys. Methods Tympanic temperatures were collected from 37 squirrel monkeys and compared to rectal temperatures using a human and veterinary infrared tympanic thermometer. Results Compared with rectal temperature measurements, the human tympanic thermometer readings were not significantly different, while the veterinary tympanic thermometer measurements were significantly higher (P < 0.05). There were no differences between sexes. Conclusions The tympanic thermometer designed for use in humans can be used in adult squirrel monkeys as an alternative to rectal thermometry for assessing core body temperature.     

Change in the staining properties of pyriform neurocytes in the mouse cerebellum during protein-calorie deficiency and subsequent rehabilitation

Quantitative analysis has been carried out on semithin sections of cerebellum cortex to investigate the relation between Purkinje cells with different dyeing properties. The number of dark Purkinje cells was found to increase after a month-long food rehabilitation of ill-fed mice. At the same time addition of carnitine to the mouse food has resulted in a significant decline in the number of dark Purkinje cells, as compared to control animals. The data obtained suggest that the rising number of dark Purkinje cells in the cerebellum cortex under conditions of malnutrition is probably due to the increased intracellular accumulation of free fatty acids.     

The effect of diet on the Vervet monkey endocrine pancreas

Vervet monkeys (Cercopithecus aethiops) used for pancreatic endocrine cell distribution studies were found to have been maintained on different diets. Although the effect of dietary changes on the exocrine pancreas has been described in several animals, little, apart from the effect of malnutrition, has been reported for the endocrine pancreas. Reported here are pancreatic endocrine cell distributions in monkeys on a standard diet (n ? 3) compared with monkeys on an atherogenic diet (n = 3). Quantitation of immunolabelled pancreatic endocrine cell types revealed a significant 80% increase in A (glucagon) cell volume in monkeys on an atherogenic diet concomitant with a significant reduction in B (insulin) cell volume to approximately 60% of normal. This reflects a pattern of events that occurs in non-insulin dependent diabetes. An accompanying reduction in PP (pancreatic polypeptide) cell volumes supports our hypothesis that altering A and PP cell volumes could reflect differential gene expression in those cells in the adult in which glucagon and PP are co-localized.