Effects of recombinant bovine interleukin-8 (rbIL-8) treatment on health, metabolism, and lactation performance in Holstein cattle I: Production and functional characterization of rbIL-8 in vitro and in vivo.

In the present study, we standardized processes of cloning and purification of recombinant bovine interleukin-8 (rbIL-8) from bacterial culture and assessed its biological activity in Holstein cattle. Plasmid containing a subclone of bovine IL-8 was expressed using Escherichia coli BL21 and cell lysate was purified by chromatography. The presence of rbIL-8 was assessed by Western blot analyses and function was confirmed in vitro using a chemotaxis chamber. Based on optical density values, chemoattractant properties of rbIL-8 were 10-fold greater compared with control wells. Two in vivo studies were conducted to assess the biological activity of rbIL8. For study 1, one-year-old Holstein heifers (n = 20) were randomly allocated to receive a single intravaginal administration containing 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 10) or a single intravaginal administration of 20 mL of saline solution (control, n = 10). For study 2, nonpregnant lactating Holstein cows (n = 31) were randomly allocated to receive an intrauterine administration with 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 11), a positive control consisting of resin-purified lysate of E. coli BL21 not transfected with the plasmid coding for rbIL-8 diluted in 20 mL of saline solution (E. coli, n = 10), and a negative control administered with 20 mL of saline solution (control, n = 10). An increase in vaginal neutrophils was observed in heifers treated with rbIL-8 within 3 h of treatment, but not in control heifers. Additionally, intrauterine administration of rbIL-8 increased the proportion of PMN cells in uterine cytological samples from 3.5% before treatment to 75.8% 24 h later-an increase that was not observed in the negative control group and cows treated with resin-purified lysate of E. coli. To further evaluate the effect of local and systemic rbIL-8 stimulation on the dynamics of circulating white blood cells, a third study was conducted. In study 3, nonpregnant 8-mo-old Holstein heifers (n = 30) were randomly allocated into 1 of 3 treatment groups: intravenous rbIL-8 (1,125 µg of rbIL-8 diluted in 5 mL of saline solution, n = 10); intravaginal rbIL-8 (1,125 µg of rbIL-8 diluted in 20 mL of saline solution; n = 10); or intravaginal saline (20 mL of saline solution, n = 10). Intravenous injection of rbIL-8 resulted in a transient increase in rectal temperature, which was greater at 2 h after treatment compared with cows treated intravaginally with rbIL-8 or heifers treated with saline solution. Heifers treated with rbIL-8 intravenously displayed a marked reduction in neutrophils, basophils, lymphocytes, and monocytes within the first 4 h posttreatment compared with heifers treated intravaginally. However, at 6 h after treatment, heifers treated with rbIL-8 intravenously displayed a rebound in white blood cell counts caused by an increase in neutrophil counts. These results show that the presented purification method is effective and results in biologically active rbIL-8 that can be used safely to modulate immune responses in cattle.

Estudo da ação da romã (Punica granatum L. ) na cicatrização de úlceras induzidas por queimadura em dorso de língua de ratos Wistar (Rattus norvegicus) / Pomegranate action study (Punica granatum L. ) for the healing of ulcers induced by burns to the dorso-lingual mucosa in Wistar rats (Rattus norvegicus).

RESUMO O objetivo desse estudo foi observar a ação da romã (Punica granatum L.) em estomatites induzidas por queimaduras no dorso da língua de ratos Wistar. Foram utilizados 24 ratos Wistar machos adultos, provenientes do Biotério da UNIVASF. Foram formados quatro grupos (G1: Polpa da romã por gavagem; G2: Polpa da romã por gavagem + aplicação local do chá da casca do fruto; G3: aplicação local do chá da casca do fruto e G4: Controle negativo). As queimaduras foram confeccionadas com instrumental odontológico padrão. Os tratamentos foram realizados duas vezes ao dia, durante 14 dias. Metade dos animais de cada grupo (n=3) foi eutanasiada no sétimo dia de experimentação, enquanto o restante foi eutanasiado no 14º dia. As línguas foram removidas e fixadas com formaldeído a 10% tamponado, processadas com cortes de 5 µm e coradas em HE. Clinicamente, os animais do grupo G2 tiveram melhores resultados. Na análise histológica qualitativa foi avaliada a reepitelização e os graus de inflamação numa escala de 0 a 4. Na análise estatística, utilizou-se o teste Qui-quadrado de Pearson. Houve significância estatística (p=0,026 e p=0,023) quando se comparou o tratamento com os graus de reepitelização e inflamação nos quatro grupos estudados. O grupo G2 apresentou cicatrização completa com 14 dias. Os piores escores obtidos foram atribuídos ao Grupo G4 nos dois parâmetros de avaliação qualitativa. Diante dos resultados obtidos, observa-se que a romã (Punica granatum L.) possui ação cicatrizante na mucosa lingual de ratos Wistar.

ABSTRACT The objective of this study was to observe the action of pomegranate (Punica granatum L.) on stomatitis induced burns on the dorso-lingual musosa in Wistar rats. Twenty-four male, adult Wister albino rats were used, from the bioterium of UNIVASF. There were four groups (G1: Pomegranate juice by gavage; G2: Pomegranate juice by gavage + local application of fruit peel tea; G3: Local application of fruit peel tea only and G4: a negative control). The burns were made with standard dental instruments. The treatments were performed twice a day for 14 days. Half the animals in each group (n = 3) were euthanized on the seventh day of experimentation, while the remainder were euthanized on day 14. The tongues were removed and fixed with a 10% formaldehyde buffer, processed as 5µm sections and stained with HE. Clinically the animals treated with tea showed better healing. For statistical analysis the Pearson chi-squared test was used. There was a statistical significance (p = 0.026 and p = 0.023) when compared to treatment with the degree of re-epithelialization and inflammation of the four groups studied. The G2 group showed complete healing within 14 days. The worst scores were found in the G4 group in both qualitative assessment parameters. Based on these results, it was observed that pomegranate (Punica granatum L.) has a healing action on the lingual mucosa of Wistar rats.

Effects of prenatal exposure to amphetamine in the medial prefrontal cortex of the rat.

This study was designed to investigate the effects of prenatal exposure to amphetamine in the organization of the medial prefrontal cortex of the rat, by an evaluation of growth, morphometric and neurochemical parameters. Pregnant Wistar rats were given 10 mg/kg body weight/day of D-amphetamine sulfate, subcutaneously, from gestational days 8 to 22. Control groups of pregnant rats were injected with saline, pair-fed or non-manipulated; litters were culled to eight pups (four males and four females), weighed every other day until postnatal day 30 and every week until day 90. The Gompertz model was used to study body weight evolution and the estimated growth parameters were not significantly different in the experimental groups. At postnatal days 14 and 30, the volumes of the prefrontal cortex, the fraction of neuropile occupied by neurons and the number of neurons per unit surface are were determined. The number of neurons per unit volume of reference area was calculated using the stereological technique of the dissector. For neurochemical analysis, the medial prefrontal cortex was dissected to measure the concentration of dopamine, serotonin and their metabolites. The allometric relationship of forebrain/body growth pointed to a mechanism of sparing and compensatory growth in the amphetamine exposed group. The changes found in the number of neurons per unit volume at postnatal day 14 show a catch-up at postnatal day 30. A decrease in serotonin levels was found in the amphetamine group compared with the pair-fed control, which was reflected in the ratio of serotonin to its metabolite, 5-hydroxyindolacetic acid. These changes, whether permanent or transitory, raise the possibility that some of the effects of prenatal exposure to amphetamine may be due to modifications in the neurotransmitter levels of serotonin.

Effects of prenatal cocaine exposure in the prefrontal cortex of the rat. A morphometric evaluation.

This work was undertaken in order to assess the organization of the prelimbic area of the medial prefrontal cortex of rats exposed prenatally to cocaine. Pregnant Wistar rats were assigned to the following groups: 1. Cocaine--60 mg/kg body wt/d sc, from gestational days 8-22; 2. Saline; 3. Pair-fed; and 4. Nonmanipulated. Male offspring were perfused on postnatal days 14 and 30. Six brains per group and per age were embedded in celloidin to calculate the volumes of the prelimbic area; sections from the other six brains were embedded in resin and processed for electron microscopy. Using semithin sections (2 microns) of layers II-III and V-VI, the following parameters were calculated: 1. The fraction of the neuropil occupied by neurons (VV); 2. The packing (NA) density; and 3. The numerical (NV) density. Qualitative alterations consisted of dispersed profiles of degenerated neurons and dendrites in the medial prefrontal cortex. No significant differences were found in the gross morphometric parameters when the cocaine group was compared with the other groups. A high interanimal variation was shown in the prelimbic volumes of postnatal day (PND) 14 cocaine-treated rats, and a a decrease in volumes was detected at PND30. Although there are some alterations in the main afferent cortical target area for dopaminergic input, its gross morphometric parameters do not seem to be sufficiently affected to account for the behavioral alterations referred to as being dependent on this brain region.

Effects of prenatal cocaine exposure on postnatal growth patterns of male Wistar rats.

The purpose of this study was to investigate basic parameters regarding the postnatal effects of prenatal exposure to cocaine. Timed-pregnant Wistar rats were injected SC with 60 mg/kg body weight/day of cocaine from gestational day 8 to 22. Control females were nonmanipulated and given food and lib; saline females received saline injections and pair-fed received saline and were nutritionally controlled to the cocaine-treated rats. Litters were restricted to 8 pups, weighed every other day until postnatal day (PND) 30 and every week from PND 30 to PND 90. The rats were perfused at PND 14, 30, and 90. The adequacy of adjustment of the logistic and Gompertz models to the body weights of the offspring was tested for the whole experimental period. The results from the Gompertz curve showed a higher growth rate and less time to reach 37% of expected mature body mass for the offspring of cocaine and pair-fed dams as compared with that of control and saline dams. No significant differences in the estimated adult weight were found among the experimental groups. The allometric relationship between forebrain and body weight is described by two postnatal growth phases with a first phase of rapid growth between PND 14 and 30 and a decelerating phase between PND 30 and 90. This relationship was not different among the experimental groups; however, the cocaine and pair-fed offspring showed a constant deficit in the forebrain weight as compared with the control and saline offspring.(ABSTRACT TRUNCATED AT 250 WORDS)