Efficacy comparison of intraperitoneal anaesthesia and post-operative analgesia regimens in laboratory rats.

INTRODUCTION: Effective and safe anesthesia for rodents has long been a leading concern among biomedical researchers. Intraperitoneal injection constitutes an alternative to inhalant anesthesia. PURPOSE: The aim of this study was to identify a safe, reliable, and effective anesthesia and postoperative analgesia protocol for laboratory rats exposed to painful procedures. MATERIAL AND METHODS: Twenty-seven female Wistar rats in an ongoing study that required surgery were randomized into groups for three different intraperitoneal anesthesia protocols and three different analgesia regimens. The anesthesia groups were (1) medetomidine + ketamine (MK), (2) ketamine + xylacine (KX), and (3) fentanyl + medetomidine (FM). Three analgesia groups were equally distributed among the anesthesia groups: (1) local mepivacaine + oral ibuprofen (MI), (2) oral tramadol + oral ibuprofen (TI), and (3) local tramadol + oral tramadol + + oral ibuprofen (TTI). A core was assigned to measure anesthesia (0-3) and analgesia (0-2) effectiveness; the lower the score, the more effective the treatment. RESULTS: The mean MK score was 0.44 versus 2.00 for FM and 2.33 for KX. Mean score for analgesia on the first postoperative day was TTI (4.66) TI (9.13), and MI (10.14). Mean score 48 hours after surgery was TTI (3.4), TI (6.71), and MI (9.5). These differences were statistically significant. CONCLUSION: MK was shown to be a reliable, safe, and effective method of anesthesia. The TTI analgesia regimen is strongly recommended in light of these results.

Is eosinophilic esophagitis an equivalent of pollen allergic asthma? Analysis of biopsies and therapy guided by component resolved diagnosis.

BACKGROUND: Eosinophilic esophagitis (EoE) is characterized by esophageal dysfunction and, histologically, by eosinophilic inflammation. There is not a clear etiologic treatment. Biopsies analysis using plant histology methods may show callose and pollen tubes in the esophageal mucosa. Component-resolved diagnosis (CRD) with microarrays could detect possible allergens involved and indicate an elimination diet and allergen immunotherapy (AIT). METHODS: One hundred and twenty-nine patients with EoE were tested for environmental and food allergens. CRD, histological and botanical analysis were performed. Clinical scores and endoscopic biopsy were performed every six months for three years. Fifty healthy patients, 50 asthmatics due to pollen, and 53 celiac disease patients were included as comparison groups. CRD-directed AIT was administered in 91 EoE patients and elimination diet in 140 patients (87 EoE and all 53 CD patients). RESULTS: CRD detected allergen hypersensitivity in 87.6% of patients with EoE. The predominant allergens were grass group 1 (55%), lipid transfer proteins (LTP) of peach and mugwort, hazelnuts and walnuts. Callose from pollen tubes was found in 65.6% of biopsies. After CRD-guided elimination diet and/or AIT, 101 (78.3%) EoE patients showed significant clinical improvement (p<0.017) and 97 (75.2%) were discharged (negative biopsy, no symptoms, no medication) without relapse. AIT-treated patients had better outcomes (odds ratio 177.3, 95% CI 16.2-1939.0). CONCLUSION: CRD-directed AIT and/or elimination diet was efficient in treating EoE patients and was well tolerated.

Adalimumab (tumor necrosis factor-blocker) reduces the expression of glial fibrillary acidic protein immunoreactivity increased by exogenous tumor necrosis factor alpha in an organotypic culture of porcine neuroretina.

PURPOSE: To determine if exogenous addition of tumor necrosis factor alpha (TNFα) exacerbates retinal reactive gliosis in an organotypic culture of porcine neuroretina and to evaluate if concomitant adalimumab, a TNF-blocker, diminishes it. METHODS: Porcine retinal explants from 20 eyeballs were cultured. Cultures with 100 pg/ml TNFα, 10 µg/ml adalimumab, 100 pg/ml TNFα plus 10 µg/ml adalimumab, or controls without additives were maintained for 9 days. Freshly detached retinas were processed in parallel. TNFα levels in control culture supernatants were quantified with enzyme-linked immunosorbent assay. Cryostat sections were doubly immunostained for glial fibrillary acidic protein (GFAP), a marker for reactive gliosis, and cellular retinaldehyde-binding protein (CRALBP), a marker for Müller cells. Sections were also labeled with the isolectin IB4, a label for microglia/macrophages. RESULTS: TNFα in control culture supernatants was detected only at day 1. Compared to the fresh neuroretinal samples, upregulation of GFAP and downregulation of CRALBP occurred during the 9 days of culture. Exogenous TNFα stimulated glial cells to upregulate GFAP and downregulate CRALBP immunoreactivity. TNFα-treated cultures also initiated the growth of gliotic membranes and underwent retinal disorganization. Adalimumab inhibited the spontaneous increases in GFAP and maintained CRALBP. In combination with TNFα, adalimumab reduced GFAP expression and conserved CRALBP, with only slight retinal disorganization. No appreciable changes in IB4 labeling were observed under the different culture conditions. CONCLUSIONS: In cultured porcine neuroretina, spontaneous reactive gliosis and retinal disorganization were exacerbated by exogenous TNFα. Adalimumab reduced spontaneous changes and those induced by TNFα. Therefore, inhibiting TNFα may represent a novel approach to controlling retinal fibrosis observed in some human diseases.

A method for obtaining Schwann cell cultures from adult rabbit nerve based on "in vitro" pre-degeneration and neuregulin treatment.

Schwann cells (SCs) are basic elements for cell therapy and tissue engineering in the central and peripheral nervous system. Therefore, the development of a reliable method to obtain SC cultures is required. For possible therapeutic applications the cultures need to produce a sufficiently large number of SCs with a high level of purity in a relatively short period of time. To increase SC yield and purity we pre-degenerated pieces of 1-2 mm of adult rabbit sciatic nerves by incubating them for seven days in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin/streptomycin and NRG1-ß1. Following pre-degeneration the nerve pieces were dissociated and then cultured for 6 or 15 days in the same culture medium. After 6 days of culture we obtained around 9.5x10³ cells/mg with approximately 94% SCs (S-100 positive) purity. After 15 days of culture the yield was about 80x10³ cells/mg and the purity was approximately 75%. Pre-degeneration and subsequent culture of small pieces of adult nerve with NRG1-ß1 supplemented medium increased the number of SCs and restricted the overgrowth of fibroblast-like cells.

Absence of relevant effects of 5 mT static magnetic field on morphology, orientation and growth of a rat Schwann cell line in culture.

The aim of this study is to observe possible changes in the morphology, orientation or cell growth of an in vitro cultured Schwann cell line by 24 h exposure to 5 mT static magnetic fields. The magnetic field generator basically consists of a pair of circular coils in a Helmholtz arrangement and enables temperature to be controlled (37+/-0.1 degrees C). We did not find any statistically significant differences in the cell growth rate between control and exposed cells, nor did we observe any differences in cell morphology or orientation.

Specific dose-dependent damage of Lieberkühn crypts promoted by large doses of type 2 ribosome-inactivating protein nigrin b intravenous injection to mice.

Nigrin b is a non-toxic type 2 ribosome-inactivating protein as active as ricin at ribosomal level but 10(5) and 5 x 10(3) times less toxic for animal cell cultures and mice, respectively, than ricin. The purpose of the present study was to analyze the effects of intravenous injection of large amounts of nigrin b to the mouse. Injection through the tail vein of 16 mg/kg body weight killed all mice studied before 2 days. Analysis of several major tissues by light microscopy did not reveal gross nigrin b-promoted changes, except in the intestines which appeared highly damaged. As a consequence of the injury, the villi and crypt structures of the small intestine disappeared, leading to profuse bleeding and death. In contrast, intravenous injection of 5 mg/kg body weight was not lethal to mice but did trigger reversible toxic effects. In both cases, lethal and sub-lethal doses, the target of nigrin b appeared to be the highly proliferating stem cells of the intestinal crypts, which had undergone apoptotic changes. In contrast to nigrin b, the injection of 3 mug/kg of ricin kills all mice in 5 days but does not trigger apoptosis in the crypts. Therefore, the effect seen with sub-lethal nigrin b concentrations seems to be specific. Nigrin b killed COLO 320 human colon adenocarcinoma cells with an IC(50) of 3.1 x 10(-8) M and the effect was parallel to the extent of DNA fragmentation of these cells. Accordingly, despite the low general toxicity exerted by nigrin b as compared with ricin, intravenous injection of large amounts of nigrin b is able to kill mouse intestinal stem cells without threatening the lives of the animals, thereby opening a door for its use for the targeting of intestinal stem cells.

Non-toxic type 2 ribosome-inactivating proteins (RIPs) from Sambucus: occurrence, cellular and molecular activities and potential uses.

Ribosome-inactivating proteins (RIPs) are a family of enzymes that trigger the catalytic inactivation of ribosomes. The most known member of the family is the highly poisonous two-chain ricin isolated from Ricinus communis L. Sambucus species contain a number of two-chain RIPs structurally and enzymatically related to ricin which have the noteworthy feature that, having an enzymatic activity on ribosomes, leading to the inhibition of protein synthesis, higher than ricin, they are lacking of the tremendous unspecific toxicity of ricin. Therefore, they have been called non-toxic type 2 RIPs. The most representative and studied members are nigrin b present in the bark of the common (black) elder Sambucus nigra L. and ebulin 1 present in the leaves of the dwarf elder Sambucus ebulus L. The molecular basis for the low unspecific activities of nigrin b and ebulin 1 as compared with ricin seems to be related with single changes of amino acids in the high affinity sugar binding sites of the B chains. These changes determine the intracellular traffic of these proteins and thus the cellular toxicity. Conjugation ofnigrin b or ebulin 1 to either transferrin or monoclonal antibodies provided highly active conjugates targeting cancer. Thus these non-toxic type 2 RIPs are promising tools for cancer therapy.

Selective calcification of rat brain lesions caused by systemic administration of kainic acid.

Dystrophic calcification of previously damaged areas of nervous tissue occurs in a wide range of human diseases. The relationship between astroglial and microglial reactions and deposits of calcium salts was studied for up to five months in rats with a brain lesion produced by systemic administration of kainate. The morphology and atomic composition of the calcium salt deposits was also studied. Two types of lesions, sclerotic and liquefactive, were observed. In sclerotic lesions hyperplasia and hypertrophy of astrocytes partially substituted for the lost neurons, reaching a maximum in about twenty-five days after treatment. In liquefactive lesions, the astrocytic reaction occurred only around the liquefactive area. Microglial reaction was similar in both types of lesion and reached its highest expression in about twenty-five days. Calcium deposits were observed in the sclerotic but not in the liquefactive lesions. Clearly distinguishable granules of calcium salts were observed in sclerotic lesions under scanning electron microscopy after only five days post-injection. The size of calcified granules increased with time reaching 40 micro m or more in diameter at five months. The atomic composition of these deposits, studied by X-ray microanalysis, showed a time-dependent increase in calcium concentration. While there was no clear relationship between astroglial and microglial reactions and calcium salt deposits, the systemic injection of kainate produced progressively larger and more concentrated calcium deposits in sclerotic, but not in liquefactive lesions.

Effects of unilateral deprivation in postnatal development of the olfactory bulb in an altricial rodent, the gerbil (Meriones unguiculatus).

To establish if olfactory bulb sensitivity to functional deprivation is related to the degree of development at birth, we studied the effects of surgical closure of one naris in the gerbil olfactory bulb development. The naris closure was performed at three different ages: at birth, P7 and P14 and maintained for 30 or 60 days. In coronal sections we measured total bulbar surface area and surface area of the different bulbar layers establishing an estimate multiple regression model for the percentage of surface area decrease in the deprived bulb related to non deprived one. The internal and external plexiform layers are the most sensitive layers to deprivation and age and duration of deprivation were factors in their mathematical models. The glomerular layer showed a surface reduction of about 25% without dependence either on age or duration. The deprived glomerular layer showed a much lower tyrosine hydroxylase-immunoreactivity and immunoreactive cell density than those in the non deprived one. However, differences in calbindin-immunoreactive and NADPH-diaphorase positive cell density between deprived and non deprived glomerular layer were not significant. Our results indicate that olfactory bulb sensitivity to functional deprivation is not related to the degree of precocity and changes in age and duration of deprivation cause different effects on the olfactory bulb layers.

Paravertebral muscles in experimental scoliosis: a light and electron microscopic study.

Experimental structural dextroconvex scoliosis was produced in rabbits by costotransversolisis with transversectomy and releasing of paravertebral muscles between TVII and TX on the right side. Two compensatory curves developed on the upper dorsal and lumbar levels. Biopsies of paravertebral muscles in experimental animals included, besides areas of normal tissue, a considerable derangement of the cell contractile apparatus with sarcoplasmic dilation and eventual cell disintegration and necrosis. Histological changes varied along levels, the convexity being more affected. The severity of changes and reduction in body weight and length were correlated with the degree of scoliosis. A selective atrophy of slow-twitch fibers was observed in experimental animals, especially at the level of the main curve, whereas fast-twitch fiber atrophy was more important caudally. Control animal biopsies always appeared normal. Our experimental model shows an overt participation of paravertebral muscles in the establishment of compensatory processes following scoliosis, although the role that paravertebral muscles play in the etiopathogenesis of human idiopathic scoliosis requires further investigation.

Prenatal development of the mammalian vomeronasal organ.

The vomeronasal organ (VNO) originates from the medial wall of the olfactory pit shortly after the middle of the embryonic period in mammals. The Anlage stage consists of a cellular bud that grows dorsally, caudally, and towards the midline leaving a groove. The following stage, Early Morphogenesis, includes the closure of the vomeronasal groove to form a parasagittal blind-ended tube in the nasal septum, which opens into the nasal and/or oral cavities. The lumen adopts a crescent shape while the epithelial lining differentiates into an increasingly wider epithelium on the concave side and a gradually thinner epithelium on the convex side. The former goes on to occupy a medial position and develops neuroblasts among supporting and undifferentiated cells, with supporting cell nuclei tending to align in the upper rows. The lateral "non-sensory" epithelium furrows, giving a kidney-shaped appearance to the VNO cross section. The next stage, Late Morphogenesis is extended up to a difference in thickness between both epithelia becomes similar to the adult, generally by birth. An increasing number of ciliary generation complexes, larger and more abundant microvilli, and an evident glycocalyx are observed in the neuroepithelium at the luminal surface, while enzymatic activities become more intense. The non-sensory epithelium appears quite mature save for its luminal surface, which is still devoid of cilia. Blood capillaries penetrate the most basal region of the neuroepithelium and vomeronasal glands are very few and immature. At birth, some neurons appear well developed to support certain functionality; however, persistence of architectural, histochemical, and ultrastructural signs of immaturity, suggests that full performance of the VNO does not occur in newborn mammals, but in prepubertal ages.

The role of retinotomy in an experimental rabbit model of proliferative vitreoretinopathy.

PURPOSE: To create a model of proliferative vitreoretinopathy (PVR) using retinotomy with vitrectomy, cryotherapy, and platelet-rich plasma (PRP) injection, which more closely resembles the human pathophysiologic condition. METHODS: One hundred and twenty albino rabbits were divided into 10 groups of 12 rabbits each and underwent the following procedures: group 1, vitrectomy; group 2, cryotherapy; group 3, PRP intravitreous injection; group 4, retinotomy; group 5, retinotomy and vitrectomy; group 6, retinotomy and cryotherapy; group 7, retinotomy and PRP injection; group 8, retinotomy, vitrectomy, and cryotherapy; group 9, vitrectomy, cryotherapy, and PRP injection and group 10, retinotomy, vitrectomy, cryotherapy, and PRP injection. All animals underwent follow-up examinations with indirect ophthalmoscopy and fundus photography on days 1, 3, 7, 10, 14, 21, and 28 after the procedure(s). Retinal changes were categorized according to the classification of Fastenberg et al. At the end of the experiments, the eyes were enucleated, and examined under light and electron microscopy. RESULTS: No retinal detachments (RDs) were observed in groups 1, 2, 4, 5, 6, and 8. RDs of varying severity were observed in group 3 (n = 1), group 7 (n = 2), group 9 (n = 6), and group 10 (n = 12). Light and transmission electron microscopy confirmed the findings. CONCLUSIONS: Combining retinotomy with vitreous removal, cryotherapy, and PRP injection creates an efficient and different model of PVR that produced RD in 100% of rabbit eyes.

Effects of perinatal administration of haloperidol on GH pituitary contents and serum levels during the first postnatal month.

Perinatal dopaminergic blockade with haloperidol caused PRL increases in rat pituitary gland and serum which persisted during the first postnatal month. However the effects of dopamine on the synthesis and secretion of GH at these early ages are unknown. With the aim of investigating the effects of this blockade on postnatal GH secretion, haloperidol (1 mg/kg i.p.) was injected daily to pregnant rats from gestational day 16 until delivery and to pups from untreated mothers between postnatal days 2-6. GH pituitary contents and serum levels were measured weekly by RIA during the first postnatal month. The results showed that haloperidol induced a long-term increase in GH pituitary contents as well as a transient increase in serum levels. The results in serum are similar to those from human neonates indicating that dopamine plays a more important role as controller of the GH secretion in newborns than in adults.

Brain lesions and water-maze learning deficits after systemic administration of kainic acid to adult rats.

The relationship between hippocampal damage and spatial learning deficiencies was studied in rats injected with kainic acid (10 mg/kg i.p.). A single injection was given either before or after the acquisition phase of the Morris water-maze task. In this acquisition phase, the animals were required to find a hidden underwater platform starting from four different points. The task was repeated twice a day for 10 days. In the retention phase after 10 days rest, the rats repeated the same task. The damage caused by the treatment occurred in several prosencephalic areas, including the piriform and enthorhinal cortices, the thalamus and the hippocampus. In the latter, greatest damage was seen in CA1 followed by CA3 while CA2 and the gyrus dentatus appeared almost unaffected. The behavioural results indicated that kainic acid impaired but did not preclude the acquisition of the water-maze task. During the retention phase, no significant differences in latencies were found between animals that were treated before and after acquisition, thus, indicating that pretraining does not play an important role in the recovery of these spatial abilities following hippocampal lesions.

Intravitreal and subretinal proliferation induced by platelet-rich plasma injection in rabbits.

We developed an experimental model of proliferative vitreoretinopathy (PVR) in albino rabbits by combining some factors suspected of causing the disease. Sixty nine eyes divided into six groups served as controls (Groups C 1-6). Forty nine eyes were divided into four experimental groups (Groups E 1-4). Group E1 (n = 12) was injected with 0.15 ml of platelet-rich plasma. In addition, Groups E2 (n = 12) and E3 (n = 12) underwent cryotherapy or vitrectomy. Group E4 (n = 13) underwent both procedures. Seven of the 13 Group 4 experimental eyes developed total retinal detachment and giant holes. None of the other groups developed more than two total retinal detachments or giant holes (P < 0.05). Light and electron microscopy showed intravitreal or preretinal proliferation composed of fibroblast-like cells. Retroretinal membranes appeared only in Group E4 eyes, composed of elongated cells with oval nuclei and abundant organelles in the cytoplasm. We believe these lesions mimic human PVR more closely than other models previously developed.

Developmental stages of the vomeronasal organ in the rat: a light and electron microscopic study.

This paper describes the development of the rat vomeronasal organ from the stage of anlage until adulthood. Groups of four rats were sacrificed daily from prenatal day 13 (E13) until birth; at days 2, 4, 7, 10, 14 and 16 after birth; weekly from day P21 to P42 plus an additional group of adults. The vomeronasal organs were processed for light microscopy, including alcian blue-PAS and NADH-diaphorase reactions, and also for electron microscopy. For summarizing our results we propose the following developmental stages: 1. Anlage (E13). 2. Early morphogenesis (E14-16). 3. Late morphogenesis (E17 to birth). 4. Initiation of secretory activity (First postnatal week). 5. Cytoarchitectural maturity (2nd postnatal week). 6. Complete maturity (From 3rd postnatal week onwards). Our results on the maturation of the histological structure and the histochemical reactions, indicate that there may be some functional activity at birth but the development of the organ still continues during the first three postnatal weeks to acquire its full functional capability.

[Morphological evaluation of the organ of Corti of guinea pigs submitted to acoustic trauma. II. Correlations of depth and surface lesions]. / Valoración morfológica del órgano de Corti del cobaya sometido a traumatismo acústico. II. Correlación en profundidad de las lesiones referidas en superficie.

Adult guinea pigs (250-500 g) were exposed to a chronic wide-band noise, at intensities ranging from 117 to 133 dB(A) at different times. While the first part of this paper concentrated on the surface study of the lesions produced by noise, this second part describes to correlating deep structural damage, based on the study of semi-thin and ultra-thin sections in the same specimen. Finally, a general discussion is presented with respect to the lesions described, both in so far as their specific characteristics as well as the possible mechanisms of damage which determine their formation.

[Morphological evaluation of the organ of Corti in the guinea pig subjected to acoustic trauma. I. Surface preparation study]. / Valoración morfológica del órgano de Corti del cobaya sometido a traumatismo acústico. I. Estudio en superficie.

Adult guinea pigs (250-500 g) were exposed to a chronic wide-band noise, at intensities ranging from 117 and 133 dB(A) at different times. The objective of this first part of the study is, after the lesions are established, to classify and put into order the structural damage produced by noise, using surface preparations observed with the light microscope and specimens prepared for scanning electron microscopy, from the organ of Corti.

Postnatal development of striatal connections in the rat: a transport study with wheat germ agglutinin-horseradish peroxidase.

This paper deals with the postnatal development of afferent and efferent connections of the rat striatum as revealed by the transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). Tracer was injected weekly from birth to the end of the first postnatal month in the head of the caudate nucleus. To control for transport from cortical areas contaminated by the micropipette, injections in newborn rats were made by either vertical or lateral penetrations. In addition some newborn and 14-day-old animals were injected only in the cortex. The results showed that at birth there was retrograde transport to the thalamus, substantia nigra and raphe nuclei. Labelling in the cortex was seen at birth but was probably due to cortical contamination. Transport from the striatum was clearly established on day 7, when a few labelled neurons were observed on both the ipsi and contralateral sides. These neurons increased in number and were distributed through layers III to VI by day 14. At this time labelled cell bodies were observed in the claustrum and lateral amygdaloid nucleus as well as in the globus pallidus and entopeduncular nucleus. On day 21 the contralateral labelling of the lateral amygdaloid nucleus was apparent. The anterograde transport from the striatum to globus pallidus, entopeduncular nucleus and substantia nigra was already visible at birth although its intensity increased during the first postnatal month.

Alterations in prolactin secretion during the 1st postnatal month following perinatal dopaminergic blockade with haloperidol.

This research was intended to study the effects of perinatal haloperidol administration on the postnatal secretion of prolactin (PRL) with the aim of investigating the existence of a 'critical period' during which the lack of dopamine influence could cause long-term alterations in the secretion of this hormone. A first group of animals, composed of pregnant rats, was injected daily with haloperidol (1 mg/kg) from day 16 of gestation to delivery. A second group of newborn rats received the same dose from days 2 to 6 after birth. Pituitary and serum PRL were measured weekly by radioimmunoassay during the 1st postnatal month in pups from the injected mothers, in postnatally injected rats, and in controls. The results showed a significant increase in the pituitary amounts of PRL that was more intense after the prenatal treatment, especially in the females. In serum, the prenatal treatment induced PRL levels higher than in the controls, whereas the postnatally injected group exhibited a V-shaped response which has been described as characteristic of neuroleptic withdrawal. These data confirm the existence of a 'critical period' during which perinatal administration of haloperidol alters the postnatal PRL production and secretion patterns. The persistence of high PRL contents in pituitary may reflect an alteration in the hormone synthesis and/or an increase in the rate of somatomammotrophes that differentiate into lactotrophes after suppression of dopamine influence. The high PRL levels in serum indicate a failure in the control of PRL release, perhaps after damaging the tuberoinfundibular neurons as a consequence of the high prolactinemia induced by the treatment.