This study addresses whether hippocampal progenitor cells express nestin following cerebral ischemia in rats. Cell counts within the hippocampal hilus were significantly greater following severe (eight-vessel occlusion) ischemia than following intermediate (four-vessel occlusion) ischemia (1527+/-87/mm2 vs. 918+/-71/mm2). Bromedeoxyuridine-positive cell counts were significantly higher with severe ischemia than with intermediate ischemia or in sham-operated animals, respectively (368+/-45, 43+/-14 and 7+/-1/mm2). In the eight-vessel occlusion group, 47+/-8/mm2 bromedeoxyuridine-labeled cells expressed nestin, significantly higher than in the four-vessel occlusion group and sham-operated animals (1+/-1 and 1+/-0/mm2, P<0.01 vs. eight-vessel occlusion, respectively). Confocal microscopy verified that a subset of the bromedeoxyuridine-positive cells expressed nestin. In conclusion, severe ischemia elicits nestin expression in hippocampal progenitor cells in rats.

An 8-vessel-occlusion (8VO) method was developed to compare with the conventional 4-vessel-occlusion (4VO) in hippocampal ischemic damage and progenitor cell induction 10 days following ischemia in female rats. Eight posture-relevant tests were performed following ischemia to correlate grades of postural abnormality with the histological outcome. The total hippocampal living cell ratio including 7 hippocampal subregions in 8VO group (n=11) was much lower than that in 4VO group (n=10, 51+/-5% vs. 78+/-4, p<0.01). In 4VO group, BrdU positive cells were mainly located in the subgranular zone (SGZ) with a count of 54+/-20/mm2 (7micro-thick slice), comparable to the maximal level following global ischemia in male gerbils and rats reported so far referring to slice-thickness differences (50-60 micro-thick slices). Similarly, nestin-bearing cells were 29+/-11/ mm2. In 8VO group, BrdU and nestin positive cells increased by 10 times. Triple staining of BrdU, nestin and DAPI demonstrated that BrdU-immunoreactivity was extensively distributed in the hippocampal hilus while the nestin was mainly located along the SGZ. Most of nestin labeling was not co-localized with the BrdU, indicating that establishment of these cells might precede BrdU injections (8 and 9d post ischemia). Behavioral scores were much greater for 8VO group than for 4VO group and composite postural scores well correlated with the hippocampal cell loss. In conclusion, severe ischemia correlates with vigorous induction of the hippocampal progenitor cells in rats while behavioral profiling of posture changes permits prediction of severity of damage.

Information is conflicting as to whether nitric oxide plays a role in the pathogenic mechanisms of zinc deficiency. Our series of research using a rat model demonstrated that inducible nitric oxide synthase in the intestine is upregulated by zinc deficiency when challenged by the injection of IL-1alpha, and the systemic administration of nitric oxide synthase inhibitor attenuates both intestinal damage and inflammatory skin lesions induced by zinc deficiency. Evidence from both transcription and translation levels indicates that inducible nitric oxide synthase, one of three nitric oxide synthase isoforms, has already been induced in the skin and intestine of zinc-deficient animals, whereas it is not generally expressed in normal tissues. On the other hand, total nitric oxide synthase activity in the intestine of zinc-deficient animals is significantly lower than that in controls, indicating that zinc deficiency may induce a potential vulnerability to nitric oxide rather than an absolute increase of nitric oxide synthase activities. Tissue zinc and metallothionein levels are significantly decreased in zinc-deficient rats, suggesting lowered antioxidative capability. Whether nitric oxide is destructive in inflammation may depend on the status of homeostasis such as the zinc level of tissues and the balance between the three nitric oxide synthase components, although identifying an absolute increase of nitric oxide production is of importance. Defining the role of nitric oxide provides the rationale for new strategies in zinc deficiency.

Skin lesions are common manifestations of zinc deficiency in humans and animals, but the pathogenic mechanisms have not been fully clarified. In the present study, a nitric oxide synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME), was given to zinc-deficient (ZD) rats to see whether it prevents or delays the occurrence of skin lesions. Weanling male rats were given free access to a ZD diet (2 mg zinc/kg) for 4 wk to induce zinc deficiency. Control rats, including pair-fed (PF) and ad libitum (AL) groups, were given a diet supplemented with zinc (50.8 mg zinc/kg. l-NAME (0.3 g/L in drinking water) was given to some ZD rats for 3 wk, starting at the second week of their ZD dieting. Dermatitis of the extremities, balanitis, stomatitis, and alopecia appeared in ZD but not in AL and PF rats. Administration of l-NAME significantly reduced the frequency of cutaneous and mucocutaneous inflammatory lesions but did not prevent alopecia in the ZD rats. Reverse transcription polymerase chain reaction showed that inducible nitric oxide synthase mRNA was expressed in the paw skin of ZD but not of AL and PF rats. Evaluation of skin microvascular permeability by the Evans blue leakage technique indicated that l-NAME administration significantly attenuated extravasation of Evans blue in the paw skin of ZD rats. Furthermore, stains positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling were condensed and diffusely distributed over the epidermis, dermis, and subcutaneous tissue of paws in ZD rats. ZD rats had intense cell infiltration and parakeratosis in the paw skin. l-NAME administration effectively prevented these morphologic changes. These results demonstrate that nitric oxide synthase inhibitor ameliorates inflammatory lesions of the skin in ZD rats.

BACKGROUND:Effect of supplemental alanyl-glutamine in standard TPN (S-TPN) on luminal mucus gel and small intestinal permeability was investigated.METHODS:Thirty Sprague-Dawley rats were divided into group I (n = 10), receiving standard rat diet; group II (n = 10), receiving S-TPN; and group III (n = 10), receiving alanyl-glutamine-supplemented TPN for 1 week. After 1 week, fluorescein isothiocyanate (FITC)-dextran was injected into the small intestine of the rats, and they were killed. A small intestinal sample and portal blood were obtained for morphologic and functional analysis of mucus gel and intestinal permeability.RESULTS:In group II, thickness and optical density of mucus gel per millimeter serosal length of intestine were significantly lower than group I (p<.001) and were significantly higher in group III than in group II (p<.001). The number of goblet cells in the villi and in the crypt of the small intestine was significantly lower in group II than in group I (p<.001) and was significantly higher in group III than in group II (p<.001), with the exception of the villi of jejunum. Villous and crypt surface area per millimeter serosal length of intestine was significantly lower in group II than in group I (p<.001) and was significantly higher in group III than in group II (p<.001). Small intestinal permeability to FITC-dextran was significantly higher in group II than in group I (p<.001) and was significantly lower in group III than in group II (p<.001). Glucosamine synthetase level was significantly higher in group III than in group I and ileum of group II (p<.001).CONCLUSIONS:Alanyl-glutamine-supplemented TPN prevents a decrease in mucus gel and an increase in small intestinal permeability associated with S-TPN.

BACKGROUNDThe effects of N-acetylcysteine (NAC) as a mucolytic agent on the uptake of fluorescent polystyrene microparticles by Peyer's patches, on intestinal permeability, and on subsequent transport to mesenteric lymph nodes (MLNs) were investigated to establish the role of mucus gel layer in this process.METHODSTwenty rats were divided into two groups: control (n = 10) and NAC (n = 10). Fluorescent polystyrene latex beads of 3.2+/-0.2 microm in diameter were used as a probe for measuring the previously mentioned parameters. The solution of latex beads (0.1 mL) was injected into a 2-cm length of ileal loop containing Peyer's patches, with 0.1 mL of saline (control group) or with 0.1 mL of NAC solution (NAC group) within 10 cm proximal from the ileocaecal valve. Intestinal loops, portal blood, and neighboring MLNs were taken within 1 hour of injection. Intestinal sections were stained by periodic acid-Schiff reagent. Peyer's patches and MLNs were analyzed for the count of particles by image analysis using a confocal laser scanning microscope.RESULTSMorphologically, periodic acid-Schiff positive uniform mucus gel was present in front of Peyer's patches of the control group, and mucus gel layer was disrupted and noncontinuous in the NAC group. The number of particles within Peyer's patches and MLNs in the NAC group was significantly higher than that in the control group (p<.001). Intestinal permeability of latex beads in the NAC group was significantly higher than that in the control group (p<.001).CONCLUSIONSThese data suggest that the mucus gel layer located in front of Peyer's patches is one of the important factors for the uptake of noxious macromolecules, and this in turn plays a major role on small intestinal permeability and subsequent translocation to MLNs.

A nitric oxide synthase (NOS) inhibitor, NG-nitro-L -arginine methyl ester (L-NAME), was given to zinc-deficient (ZD) rats to determine whether it prevents the intestinal damage usually observed under these conditions. Weanling male rats were given free access to a ZD diet (2 mg zinc/kg), whereas control rats including pair-fed (PF) and ad libitum consumption (AL) groups were given a zinc-supplemented (50.8 mg zinc/kg) diet for 4 wk. Half of the ZD rats received L-NAME (0.3 g/L in drinking water) for 3 wk starting at the wk 2 of the deficient period. Plasma zinc concentration in ZD rats was significantly lower (P < 0.05) than that of AL and PF rats. Administration of L-NAME did not alter this concentration. Intestinal zinc concentration did not differ among groups. However, metallothionein-1 (MT-1) mRNA level was significantly lower in the intestine of ZD rats than in AL or PF rats. Treatment of ZD rats with L-NAME did not affect this level. Intestinal microvascular permeability evaluated by Evans blue showed significantly higher extravasation in ZD rats than in AL rats, whereas L-NAME administration inhibited the extravasation. Expression of inducible NOS mRNA was observed in intestine of ZD but not of AL or PF rats, and there was no significant difference between ZD rats, regardless of L-NAME treatment. The activity ratio of inducible NOS to total NOS in ZD rats not receiving L-NAME was significantly higher than that in AL rats or ZD rats treated with L-NAME (P < 0.05). The number of apoptotic-positive and goblet cells in intestinal villi was significantly higher in ZD rats compared with AL or PF rats. L-NAME administration in ZD rats reversed this effect. These results indicate that inhibition of NOS ameliorates zinc deficiency-induced intestinal damage in rats.

This study investigated whether interleukin-1alpha-induced metallothionein gene expression is affected by zinc deficiency. Weaning male rats were fed a zinc-deficient (ZD) diet (2 mg zinc/kg) or a zinc-supplemented diet [50.8 mg zinc/kg; controls for the diet included pair-fed (PF) and ad libitum consumption groups (AL)] for 4 wk. All rats except those that served as controls for interleukin-1alpha administration, (injected with vehicle and killed at 0 h) were then injected subcutaneously with interleukin-1alpha (2 x 10(7) units/kg body wt) and killed at 3, 6, 12, 24 and 72 h after the injection. Compared with AL and/or PF rats, zinc depletion significantly reduced zinc concentrations in plasma and liver but not in kidney or intestine, and significantly reduced hepatic, renal, and intestinal metallothionein-1 mRNA levels analyzed by competitive reverse transcription-polymerase chain reaction (RT-PCR). Interleukin-1alpha injection reduced plasma zinc concentration and enhanced liver zinc concentration, but did not affect zinc levels in kidney or intestine. Metallothionein-1 mRNA was significantly elevated by interleukin-1alpha in liver, kidney and intestine of all groups; the levels in liver and kidney of ZD rats 6 h after the injection were significantly higher than those of AL or PF rats. Liver metallothionein protein levels were enhanced after interleukin-1alpha injection in both AL and ZD rats. Semiquantitative RT-PCR revealed significantly higher hepatic levels of interleukin-1 receptor type-I mRNA in ZD rats than in AL and PF rats but no differences in renal or intestinal tissues among groups before interleukin-1alpha challenge. In conclusion, zinc deficiency induces upregulation of metallothionein-1 gene expression in response to interleukin-1alpha challenge in rats.

BACKGROUND Dysfunction of the intestinal barrier, as evidenced by increased intestinal permeability and bacterial translocation, has been reported under total parenteral nutrition (TPN). However, the role of Peyer's patches on the intestinal barrier in TPN is not well understood. We investigated whether TPN alters the uptake of microparticles by the follicle-associated epithelium of Peyer's patches. METHODS Twenty rats were divided into two groups, a control group and a TPN group. Fluorescent polystyrene latex beads, 3.2 +/- 0.2 microns in diameter, were used as a probe for measuring the uptake by Peyer's patches. After 1 week of consuming either the control or TPN diet, rats were killed. On the day of killing, 0.1 mL of latex beads solution was injected into a 1-cm length of ileal loop, within 10 cm of the ileocecal valve. Samples were taken after 30 minutes of injection, sectioned by cryostat, and then viewed under a fluorescent microscope. Follicle-associated epithelial length and particles were counted using a confocal laser scanning microscope. The number of particles within each compartment was standardized per unit length of epithelium of Peyer's patches. RESULTS Particle numbers within Peyer's patch dome of the TPN group were significantly increased compared with those of the control group (p < .01). CONCLUSIONS These data suggest that dysfunction of the intestinal barrier in TPN might be associated with a change of uptake by Peyer's patches.

Synthesis of inducible nitric oxide synthase (iNOS) in the intestine may result in local tissue damage. We investigated whether a challenge with interleukin-1alpha could give rise to intestinal iNOS expression and diarrhea in rats of differing zinc status. Weaning male rats were fed a zinc-deficient (ZD) diet (2 mg zinc/kg) for 4 wk to induce zinc deficiency or a zinc-supplemented diet [50.8 mg zinc/kg; controls, including pair-fed (PF ) and ad libitum (AL) consumption groups], and then subcutaneously injected with interleukin-1alpha (2 x 10(7) units/kg body wt). Without the interleukin-1alpha challenge, ZD rats had significantly lower plasma zinc concentration than the other groups. Intestinal metallothionein-1 mRNA abundance was lower in ZD rats than in AL rats. iNOS was expressed in the intestine of ZD rats but not in the others. None of the rats experienced diarrhea during the feeding period. Interleukin-1alpha led to a reduction in plasma zinc concentration, enhancement in intestinal metallothionein-1 mRNA levels, and expression of the intestinal iNOS gene in all groups. However, the abundance of iNOS mRNA was significantly higher in ZD rats than in the other groups. The presence of iNOS protein was demonstrated by immunohistochemical staining in the intestine of ZD rats that had been treated with interleukin-1alpha 12 h earlier. In addition, diarrhea occurred in most of the ZD rats and some of the PF rats but not in AL rats after interleukin-1alpha treatment. We conclude that ZD rats respond to interleukin-1alpha challenge more severely than controls, reflected by a more marked and prolonged iNOS expression and a greater incidence of diarrhea.

In view of the potential relationship between cerebral ischemia and zinc status, this study was designed to examine whether dietary zinc-deficiency would affect the outcome of focal cerebral ischemia in rats. Forty-five male Sprague-Dowley rats were randomly divided into five groups: (1) eight rats received a semi-purified zinc-deficient diet containing less than 0.38mg/100g of zinc for 1 week (ZD-1w); (2) eleven rats received the same diet as described as above for 2 weeks (ZD-2w); (3) ten rats, named pair-fed control group (PF-2w), received a zinc-supplemented diet and matched by the amount of diet equal to that consumed by ZD-2w group for 2 weeks; (4) ad libitum-fed control group received the same diet as taken by PF-2w animals for 1 week (AF-1w, n=6) and (5) the remaining 10 rats were named as no-fed (NF) group, in which the rats were directly subjected to cerebral ischemia without entering the feeding process. After experiencing the dieting stage for 1 or 2 weeks, or without (NF group), respectively, the animals were subjected to permanent occlusion of the middle cerebral artery (MCA). Plasma zinc concentration and the cerebral infarct volume were determined at 24 hours of ischemia. The final level of zinc in NF and AF-1w rats was 139±5 and 127±6 μg/100 ml, respectively. The zinc-deficient diet reduced the concentration by 52% in ZD-1w and by 64% in ZD-2w group. The increment in infarct size in ZD-1w group was inconspicuous. However, treatment with zinc-deficient diet for 2 weeks increased the total infarct volume by 118%, 88%, 58% and 47% as compared with NF, AF-1w, PF-2w and ZD-1w rats, respectively. Especially, the cortical infarct volume (206±35 mm3) in ZD-2w group was 98–227% larger than those in the other groups (p<0.05, respectively). In conclusion, dietary zinc-deficiency increases infarct size following permanent MCA occlusion in rats. In addition, the aggravation of cerebral ischemia may depend on the severity of zinc-deficiency, duration and the timing of dietary restriction.

OBJECTIVEThe authors determined the effects of alanyl-glutamine-supplemented total parenteral nutrition (TPN) on mucosal metabolism, integrity, and permeability of the small intestine in rats.METHODSMale Sprague-Dawley rats were randomized to receive TPN supplemented with a conventional amino acids mixture (STD group) or the same solution supplemented with alanyl-glutamine; both solutions were isocaloric and isonitrogenous. On the seventh day of TPN, D-xylose and fluorescein isothiocyanate (FITC)-dextran were administered orally. One hour later, superior mesenteric vein (SMV) D-xylose and plasma FITC-dextran concentration were measured. Intestinal blood flow and calculated intestinal substrates flux were measured with ultrasonic transit time flowmetery.RESULTSPlasma FITC-dextran increased significantly in the STD group. Intestinal blood flow and SMV D-xylose concentration did not differ between the groups. Mucosa weight, villus height, mucosal wall thickness, mucosal protein, and DNA and RNA content in jejunal mucosa were significantly increased in the alanyl-glutamine group. Jejunal mucosal glutaminase activity and net intestinal uptake of glutamine (glutamine flux) were significantly higher in the alanyl-glutamine group as compared with the STD group.CONCLUSIONAddition of alanyl-glutamine dipeptide to the TPN solution improves intestinal glutamine metabolism and prevents mucosal atrophy and deterioration of permeability.

BACKGROUNDFrom the developmental aspects, the distribution of fluorescein isothiocyanate dextran 70,000 (FTTC-dextran) and mucous gel across the lumen of small intestine was observed as an investigation into the role of mucous gel on intestinal permeability. Furthermore, the effect of N-acetyl cysteine (NAC), a mucolytic agent, on intestinal permeability was examined.METHODSIn suckling and weaned rats, FTTC-dextran (750 mg/kg body wt) was gavage-fed. After 3 hours, blood samples were taken by cardiac puncture to analyze plasma FTTC-dextran by fluorescence spectrometry. Samples of small intestine with luminal contents were frozen and sectioned in a cryostat for fluorescence microscopy; the same sections were placed in a 0.2% celloidin solution to preserve mucous gel and were stained by periodic acid-Schiff reaction for light microscopy. In weaned rats, intestinal permeability was examined with different concentrations of intraluminally instilled NAC.RESULTSThe plasma level of FTTC-dextran showed a significant increase (p < .01) in suckling rats compared with the weaned rats. Morphologic findings were similar in both the jejunum and ileum: The spaces between villi were not entirely filled with mucus but filled with FTTC-dextran in suckling rats, whereas the spaces were filled with mucus and not filled with FTTC-dextran in weaned rats. Intestinal permeability in groups with NAC were significantly higher (p < .01) than that in group without NAC.CONCLUSIONSThese results suggest that an increase in the mucous gel layer that coats the epithelial lining according to the maturation of the gastrointestinal tract is one of the most important factors for a restriction in intestinal permeability.

BACKGROUND Although it has been reported that total parenteral nutrition induces an increased intestinal permeability and a decreased mucous gel layer covering the intestinal epithelium, the role of mucous gel on intestinal permeability has not been well understood. We examined the in vivo effects of N-acetyl cysteine (NAC) as mucolytic agent and colchicine as suppressant of the mucus production on the intestinal transmission of fluorescein isothiocyanate dextran 70,000 (FITC-dextran). METHODS Rats were divided into four groups. In each group, FITC-dextran (750 mg/kg) with or without NAC (3000 mg/kg) was injected into the small intestinal lumen 3 hours after intraperitoneal injection of saline or colchicine (Col, 10 mg/kg). Thirty minutes after injection of FITC-dextran, blood samples were taken from portal vein to analyze plasma fluorescein concentration by fluorescence spectrometry. Samples of small intestine were sectioned in a cryostat for fluorescence microscopy, and the identical sections were stained by periodic acid-Schiff reaction. RESULTS Plasma FITC-dextran level in NAC group was higher than that in control group (p < .01), that in Col + NAC group was higher than that in Col group (p < .01) and that in Col + NAC group was higher than that in NAC group (p < .05). The spaces between villi were filled with mucous gel in the control and Col groups, whereas those were not entirely filled with mucous gel in NAC and Col + NAC groups. FITC-dextran and mucous gel showed complementary distribution in all rats. The villous interstitial edema was recognized in NAC group and the villi were disrupted in Col + NAC group. CONCLUSIONS These results suggest that intestinal permeability is possibly affected not only by the mucous gel covering the intestinal epithelium but also by mucus release from goblet cells of the small intestine.

Effects of zinc deficiency on intestinal disaccharidase remain controversial. This study investigated the effects of a prolonged zinc-deficient diet on selected intestinal enzyme activities and morphology. Twenty-four Sprague-Dawley rats were randomly divided into zinc-deficient, pair-fed, and ad libitum control group and fed respective diet for 7 weeks. Alkaline phosphatase and disaccharidase (maltase, sucrase, lactase) activities in jejunal and ileal mucosa as well as in feces were measured. Morphological changes were examined under the microscope at the end of the experiment. The activity of intestinal alkaline phosphatase was lower in zinc-deficient and pair-fed groups than that in ad libitum control group. Time-dependent decrease in activity of alkaline phosphatase in feces was more apparent in zinc-deficient group than that in the other two groups. The maltase and sucrase activities decreased in jejunal but not in ileal mucosa in zinc-deficient group. The maltase activity in feces decreased during the final 2 weeks of the experiment, but the sucrase and lactase activities did not in zinc-deficient group. The activity of disaccharidase in intestinal mucosa and in feces did not decrease (even increased in jejunal mucosa), although zinc content in the tissues or the excretion slightly decreased in pair-fed control group. The decreased villus height and crypt depth, flawed brush border were observed in jejunum and ileum of zinc deficient group. The data indicate that administration of zinc-deficient diet resulted in decreased activity of alkaline phosphatase and maltase and induced morphological changes in the intestine and time-related decrease of maltase activity in feces of rats. (C) 1996 Wiley-Liss, Inc.