Primary C cell hyperplasia of the thyroid in Fischer 344 rats. An immunohistochemical study

C cell proliferation of the thyroid, which was designated as primary C cell hyperplasia (PCCH), was demonstrated in aged Fischer 344 rats in high incidence using the immunohistochemical method for calcitonin. It developed from mild to severe PCCH and resulted in nodular PCCH and tumor formation. The combined incidence of PCCH and nodular PCCH was increased with age and appeared in 60.7% of 7-15 month old group and 92.7% of 16-24 month old group possibly as a preneoplastic C cell lesion. It is almost an equivalent C cell lesion reported in the human thyroid with familial C cell carcinoma and therefore this Fischer 344 rat can provide a useful animal model to study familial C cell tumor of the thyroid.     

Vasoactive intestinal peptide in the human pituitary gland and adenomas. An immunocytochemical study

Recent evidence indicates that vasoactive intestinal peptide (VIP) may be involved in normal pituitary function. Immunocytochemistry was used to localize VIP in human biopsied pituitary adenomas and postmortem anterior pituitary glands. Paraffin sections were immunostained for VIP with the avidin-biotin-peroxidase technique. Strong VIP-like immunoreactivity (VIP-LI) was observed in 16 of 17 prolactinomas, 12 of 14 growth hormone-secreting tumors associated with acromegaly, four of 12 ACTH-secreting tumors, and 14 of 18 nonfunctioning pituitary adenomas. In most cases, VIP was colocalized with the classical pituitary hormones. Six of the 18 nonfunctioning tumors had no demonstrable hormone immunoreactivity; five of these stained strongly for VIP, whereas one was negative. Of 18 normal anterior pituitaries, 12 showed strong diffuse staining for VIP throughout the gland. One pituitary with VIP-LI came from an individual who had undergone pituitary stalk transection. Double-immunoenzyme labeling and immunoelectron microscopy demonstrated VIP-LI in many lactotrophs, scattered thyrotrophs, corticotrophs, and in an occasional gonadotroph. These results suggest the following: 1) VIP is present in more than one cell type in normal and adenomatous human pituitaries; and 2) VIP may be involved in the function and development of pituitary tumors.     

Normal LHRH neuronal function and hyperprolactinemia in old pseudopregnant Fischer 344 rats

The relative contributions of LHRH neuronal function and hyperprolactinemia to the maintenance of the repeated pseudopregnant (PP) state in old Fischer 344 rats were examined. LHRH concentrations within 8 microdissected regions of the preoptico-tuberal pathway were not different between normally cycling 4-5 and 10-11 months old rats and PP 22-23 months old rats. LHRH concentrations were significantly decreased in the median eminence and serum LH was increased 2 weeks after ovariectomy in all 3 age groups. Ovariectomy had no significant effect on LHRH concentrations in any of the other brain regions examined in these three age-groups. Serum prolactin levels were elevated 4-fold in old PP rats when compared to younger animals and these old PP rats failed to exhibit the normally observed decline in serum prolactin in response to ovariectomy. Daily treatment for 10 days with the dopamine agonist, CB-154, reinitiated normal ovarian cycles in 7 of 7 old PP rats while vehicle treatment was ineffective in altering the PP state. The data suggest that persistent hyperprolactinemia rather than impaired LH secretory mechanisms is primarily responsible for the PP state in old Fischer 344 rats.     

Activation-induced apoptosis in T cells from young and old Fischer 344 rats

BACKGROUND: Activation-induced apoptosis is believed to limit cell proliferation and eliminate the high number of activated cells during an immune response. METHODS: Activation-induced apoptosis was investigated in splenic T cells isolated from young (6 months) and old (24 months) male Fischer 344 rats. The cells were incubated with anti-CD3, concanavalin A or staphylococcal enterotoxin B (primary stimulus) for 72 h, followed by restimulation with anti-CD3 or concanavalin A (secondary stimulus). Apoptosis was assessed by DNA fragmentation assay and DNA gel electrophoresis. The expression of the apoptotic marker CD95 was analyzed by flow cytometry and the relative levels of CD95 ligand, Bcl-2 and Bax protein were measured by immunoblotting. RESULTS: It was shown that DNA fragmentation was very low in the unstimulated T cells from both young and old rats. However, the level of DNA fragmentation was 45-55% greater in the activated T cells from old rats than in the activated T cells from young rats. The increase in DNA fragmentation was paralleled by an increase in the proportion of cells expressing the CD95 molecule. The proportion of CD95+ cells was approximately 40% higher in T cells from old rats than in T cells from young rats. In addition, it was found that the expression of CD95 ligand and Bax increased and Bcl-2 decreased in the activated T cells from old rats compared to the activated T cells from young rats. CONCLUSION: Our data suggest that the increase in sensitivity of T cells to apoptosis with age may contribute to age-associated immune dysfunction and disorders.     

Mediation of reflex tachycardia becomes exclusively beta-adrenergic in old Fischer 344 rats

To study how baroreflex regulation changes with age we compared reflex heart rate responses elicited in awake Fischer 344 rats of different ages during intravenous infusions of phenylephrine or sodium nitroprusside. Underlying neural mechanisms were assessed by repeating baroreflex tests following cholinergic blockade with methylatropine or beta-adrenergic blockade with propranolol. Basal heart rates always tended to be lower in old than in young rats, but the differences became statistically significant only after beta-adrenergic or combined cholinergic and beta-adrenergic blockade. Most reflex heart rate responses (i.e. except reflex tachycardia in males during depressor responses to sodium nitroprusside) were initially weaker in old than in younger rats. Reflex bradycardia and tachycardia were reduced equally in both age groups after cholinergic blockade, but were reduced more in old than in young rats after beta-adrenergic blockade. beta-adrenergic blockade alone reduced reflex tachycardia as much as did combined blockade thereby suggesting that the predominant neural mechanism was beta-adrenergic. Taken altogether these results are compatible with the interpretation that efferent pathways for mediating heart rate reflexes in Fischer 344 rats are altered with age such that as parasympathetic mediation diminishes, residual mediation particularly of reflex tachycardia, becomes almost exclusively beta-adrenergic or sympathetic.     

Vasopressin in aged rats: longitudinal studies of vasopressin excretion in Sprague-Dawley and Fischer 344 strains

In order to provide physiological baseline values for future experimental procedures, indices of vasopressin secretion were assessed in male Sprague-Dawley (SD) and Fischer 344 (F344) rats at 3 and 20 months of age. Daily water intake, urine volume, urine osmolality, and urinary vasopressin excretion were monitored in SD rats for 30 days, and in F344 rats for 60 days. In the SD strain, daily water and urine volumes, expressed as ml/24 hr/100 g b.wt., were consistently lower in aged animals, as was a calculation of water balance (water intake-urine output volumes/24 hr). Although mean VP concentration in urine appeared higher in aged rats (33.9 +/- 20.4 pg/ml) than in young (16.3 +/- 7.7 pg/ml), total daily VP excretion was comparable for both ages when expressed as a function of body weight [80.6 +/- 37.3 pg for 3 months old (m.o.) and 81.9 +/- 47.2 pg/24 hr/100 g b.wt. for 3 and 20 m.o. respectively]. Young and old F344 males showed comparable daily drinking and urine volumes, and water balance, during two months of monitoring, but VP excretion was lower (p less than 0.025) in aged rats (83.8 +/- 19.0 pg/24 hr/100 g b.wt.) than in 3 m.o. rats (213.0 +/- 48.1 pg/24 hr/100 g b.wt.). Urine VP concentration was comparable (69.6 +/- 20.6 for 3 m.o.; 59.8 +/- 25.6 pg/ml for 20 m.o.). Mean urine osmolality was not significantly different among groups. Urine osmolality and daily urine volumes showed a significant correlation with daily VP excretion among young, but not aged, rats of both strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

An immunocytochemical study of human pituitary mammotropes from fetal life to old age

The objectives were to (a) describe the cytology and distribution of mammotropes in the human pituitary gland, (b) determine whether the mammotrope is a distinctive secretory cell type and (c) ascertain when it first appears in the fetal hypophysis. Identification of mammotropes was based primarily on the Sternberger peroxidase-antiperoxidase immunocytochemical method used with an antiserum to human prolactin. Hypophyses from 25 male and 6 female adults, and 21 fetuses ranging in gestational age from 6 to 23 weeks were studied. In the adult two morphological forms of mammotropes were observed. Mammotrope I possessed a small perikaryon that commonly was located centrally in parenchymal cell cords. From the perikaryon long cytoplasmic processes extended toward neighboring capillaries. Mammotrope I reached its highest incidence in the posterolateral zones of the pars distalis. Mammotrope II possessed a larger perikaryon with short processes; cells of this form were fewer and occurred chiefly in the anteromedian zone. Mammotropes with intermediate morphological features that prevented classification into categories I or II were common in some hypophyses. Both forms of mammotropes were present prepuberally (one 6-week and one 9-year-old male) and in adult males and females. Mammotropes were only slightly more prominent in females than males. Regression of mammotropes was evident in old age. Mammotropes were distinctly different from somatotropes, corticotropes, gonadotropes and thyrotropes. In the fetal hypophysis mammotropes appeared first at 14 weeks of gestational age and remained few through 16.5 weeks. Their number increased greatly at 23 weeks.     

Neurochemical, endocrine and immunological responses to stress in young and old Fischer 344 male rats

Two experiments were performed. In the first, a 20 min conditioned emotional response (CER) paradigm was used to compare the neurochemical, endocrine and immunological responses to stress of 7- and 22-month-old Fischer 344 (F344) male rats. In the second, corticosterone levels 20 min following ether stress, and regional brain type I and II corticosterone receptor densities were examined using 7- and 17.5-month-old F344 male rats. Dopamine (DA) metabolism in old nonstressed rats was significantly reduced in the medial frontal cortex, neostriatum, nucleus accumbens and hypothalamus, but not in the amygdala. The CER procedure, nevertheless, increased medial frontal cortical, nucleus accumbens and amygdaloid DA turnover in both the young and old rats. The young and old nonstressed rats did not evidence differences in norepinephrine (NE) and serotonin (5-HT) concentrations. However, stress resulted in a decrease in medial frontal cortical 5-hydroxyindoleacetic acid (5-HIAA) and hypothalamic 5-HT levels in old but not in young animals. These observations suggest age-related differences in the response of central NE and 5-HT systems to stress. Ether and the CER procedure led to exaggerated corticosterone responses in the old rats (17.5 and 22 month, respectively). Hippocampal type I but not type II corticosterone receptors were decreased by 47% in the 17.5-month-old rats. Thus, age-related changes in hippocampal corticosterone receptor types do not occur in unison, and the exacerbated corticosterone response to stress precedes the reported down-regulation of hippocampal type II corticosterone receptors in aged rats. Age-related changes were not observed in the concentrations of corticosterone receptors in other brain regions, or in the prolactin response to stress. The old rats, however, evidenced a reduction in the availability of the renin substrate, angiotensinogen, and in stress-induced renin secretion. Immune function was impaired in the old nonstressed rats, and further compromised by exposure to the CER procedure. In comparison to the young control rats, the old nonstressed rats showed an increased percentage of splenic large granular lymphocytes, reduced splenic natural killer cytotoxicity, and impaired Con-A-stimulated splenic T lymphocyte proliferation. Reductions in T splenic cell proliferation and natural killer cytotoxicity were observed in the young rats subjected to the CER paradigm, but not to the same extent as in the old rats. These observations indicate that aging male F344 rats evidence major alterations in basal central monoamine, endocrine and immune functions, and an increased sensitivity of these systems to stress.     

A comparison of skeletal muscle morphology with training between young and old Fischer 344 rats

Muscle mass, fiber area, total fiber number, fiber population and capillarity were assessed in 6- and 25-month-old animals trained by treadmill running at 75% mean maximal capacity for 10 weeks. Serial cross-sections were stained for ATPase activity to differentiate fiber types and expose capillaries. Aging and training effects were demonstrated in maximal running speed and endurance running time. Soleus muscle mass increased in the aged, however, soleus and EDL total fiber number were declining. When adjusted for muscle weight, a significant reduction existed in fiber number for the aged soleus. Fiber area increased in the old soleus compared to young. All changes in the EDL were specific to the deep region. While there was a tendency for the capillaries/fiber and fiber area to be higher with training in the Type I fibers of the soleus, it was only significant for the young animals. Thus, while the majority of variables that showed a training effect did so across both age groups, this was not the case for vascularity. Age-associated changes in muscle morphology appear to be both muscle and fiber specific.     

Hippocampal neuronal necrosis in control Fischer 344 rats

Brain tissue sections from control male and female Fischer 344 (F344) rats from ten National Toxicology Program (NTP) Bioassays were histomorphologically reviewed for non-neoplastic lesions of the hippocampus. Unilateral segmental hippocampal neuronal necrosis, which has not been reported in normal rats, was observed in 9 of 433 (2.1 per cent) males and 1 of 454 (0.2 per cent) females. Significant coexisting lesions were left-sided atrial and/or valvular thrombosis, metastatic mesothelioma, and large lymphocyte leukaemia. These data suggest that this naturally occurring lesion of predominantly aged male rats may result from an impairment of cerebral perfusion secondary to vascular obstruction by thrombotic emboli or leukaemic cells and haemolytic anaemia concomitant with large lymphocyte leukaemia, which commonly occurs in F344 rats.     

Inhibition of estrogen-induced pituitary tumor growth and angiogenesis in Fischer 344 rats by the matrix metalloproteinase inhibitor batimastat

The development of estrogen-induced pituitary prolactinoma in Fischer 344 (F344) rats is associated with enhanced neovascularization. Based on the significance of matrix metalloproteinases (MMPs) for tumor growth and angiogenesis, we have studied the effect of batimastat (BB-94), a synthetic MMPs inhibitor (MMPI) on the progression of prolactin-secreting pituitary adenoma in rats. Pituitary tumors were induced in male F344 rats by s.c. implantation of Silastic tubes containing diethylstilbestrol (DES). The effects of chronic treatment with BB-94 (30 mg/kg b.w.) on pituitary weight, cell proliferation, apoptosis and vascular density were evaluated. We have stated that chronic treatment with batimastat caused a significant reduction in the pituitary weight. Batimastat has been found to decrease cell proliferation evaluated by a number of PCNA-positive stained cell nuclei. A marked increase in the apoptotic index within the pituitary was observed in the study group. Moreover, the density of microvessels identified by CD31 was reduced in the group treated with BB-94. The results of our study provide evidence for an inhibitory effect of batimastat, a synthetic MMPI, on the growth and angiogenesis in an experimental model of human prolactinoma. The ability of BB-94 to suppress established pituitary tumor growth suggests a possible application of MMPIs in the treatment of pituitary adenomas.     

Null cell adenomas,oncocytomas, and gonadotroph adenomas of the human pituitary: An immunocytochemical and ultrastructural anafysis of 300 cases

The immunocytochemical profile of 300 clinically nonsecreting pituitary adenomas was investigated. All tumors were diagnosed, classified, and separated into null cell adenomas, oncocytomas, and gonadotroph adenomas according to their ultrastructural morphology. The immunocytochemical analysis was based on the semiquantitative proportional estimates of positive cells immunostained for all known peptide and glycoprotein pituitary hormones including alpha-subunit. The majority of tumors (87%) were to some extent immunopositive for various hormones. Glycoprotein hormones were most frequently encountered. Usually, particularly in males, more than one subunit was present in the same tumor. In 97 tumors (32%) more than 25% of adenoma cells were immunoreactive for gfycoprotein hormones. Fifty-five tumors (18%) contained occasional cells immunopositive for growth hormone (GH), prolactin (PRL), and adenocorticotropin (ACTH) in addition to glycoprotein hormones. Given the significant proportion of immunoreactive cells for gonadotropins and alpha-subunit, in tumors characterizedas null cell adenomas and oncocytomas, imrnunocytochemistry may provide valuable information to the pathologist and clinical endocrinologist contributing to the evaluation of this heterogeneous group of tumors.     

Effects of subchronic d-fenfluramine on splenic immune functions in young and old male and female Fischer 344 rats.

The present study was designed to demonstrate age- and sex-related differences in immune functions, and to determine whether subchronic elevations in serotonin (5-HT) availability in vivo would alter immune functions assessed subsequently in vitro. Male and female F344 rats (5 and 21 months of age) were administered the 5-HT releaser and reuptake inhibitor, d-fenfluramine (d-Fen), in their drinking water for 30-38 days then killed. The young animals received a higher dose (1.8 mg/kg/day) of d-Fen than the old rats (0.6 mg/kg/day) in order to compensate for age-related decreases in drug biotransformation and clearance. Brain and spleen d-Fen and metabolite concentrations, however, were considerably higher in the young than in the old rats. d-Fen treatment did not affect body weight or fluid intake. Although substantial sex differences in immune function were not discerned, age-related decreases were observed in absolute splenic cellularity, recombinant interleukin-2 (rIL-2) stimulated natural killer (NK) cytotoxicity, LPS stimulated B-cell mitogenesis, and in the level of Ox19 (CD5) positive cells. d-Fen caused an increase in absolute spleen weight and a decrease in absolute splenic cellularity only in the old rats of both sexes. Spleen cells from young male and old female rats receiving d-Fen had relatively more large granular lymphocytes and enhanced baseline and rIL-2 activated killing of YAC-1 cells than their vehicle matched or opposite sex counterparts. The drug also increased Con A-induced T-cell proliferation in young males and LPS induced B-cell proliferation in old females. d-Fen decreased Ox39 (CD25) levels by 19%, but did not affect any of the other phenotypes examined. The results suggest that 5-HT has a selective stimulatory effect on young male and old female NK activity, and that old female rats are more sensitive to the immunological effects of d-Fen than old male rats.     

An autoradiographic and immunocytochemical study of the neonatal rat pituitary gland

Two-day-old female rats were injected with 5 nmole/kg of 6,7-³H-11β-methoxy-17-ethylestradiol (R 2858 = moxestrol) and killed one hour later. The animals were decapitated and, the pituitary glands were removed, mounted on tissue holders and frozen in liquified propane. The tissue was then processed for autoradiography according to the thaw mount technique. At the end of the exposure time, prior to photographic development, some of the tissue was fixed in 10% formalin and then photographically developed for autoradiography. The fixed tissue was subsequently stained immunocytochemically using antibodies to luteinizing hormone or prolactin. Between 10 and 15% of the cells of the pars distalis concentrated the synthetic estrogen or its metabolite. The immunocytochemical procedure revealed that both LH-gonadotrophs and lactotrophs concentrated the steroid. These studies along with earlier studies suggest that the neonatal rat pituitary contains only a small portion of the adult complement of estrogen receptors and that these receptors are dispersed across a number of cell types.     

Decreased sympathetic innervation of spleen in aged Fischer 344 rats

Splenic noradrenergic innervation in young adult and aged Fischer 344 rats was examined using fluorescence histochemistry for catecholamines and high performance liquid chromatography with electrochemical detection (LCEC) for the quantitation of norepinephrine (NE). In young adult rats, abundant noradrenergic plexuses followed the vasculature and trabeculae into splenic white pulp. In aged rats, noradrenergic innervation was reduced in density and in overall intensity of fluorescence, and splenic NE levels were significantly lower. The relationship between diminished noradrenergic innervation and diminished immune responsiveness in aging mammals, while not clear on a causal level, is presented as a hypothesis for further testing.     

Differences in the sensitivity of Fischer and Sprague-Dawley rats to aminoglycoside nephrotoxicity

Because of the anecdotal but unconfirmed inferences of the greater sensitivity of Fischer rats to aminoglycoside nephrotoxicity, an intrastudy comparison of the sensitivity of Fischer and Sprague-Dawley rats to aminoglycoside nephrotoxicity was undertaken. Tobramycin was administered at 3 dose levels to each strain of rat for 10 days. Nephrotoxicity was evaluated utilizing a spectrum of both functional and morphologic assessments of renal proximal tubular integrity. The results confirm that the aged matched Fischer rat is more sensitive to the nephrotoxic effect of tobramycin than the Sprague-Dawley. These results also suggest an opportunity to study quantitative and perhaps qualitative differences in pathogenic mechanisms of aminoglycoside nephrotoxicity in a single laboratory animal species.     

Basal and carrageenan-induced pain behavior in Sprague-Dawley, Lewis and Fischer rats

Individual differences in pain sensitivity are believed to reflect the interplay of many factors, including genetics. Inbred rat strains can be used to study the impact of genetic factors on pain sensitivity. Inbred Lewis (LEW) and Fischer 344 (FIS) rat strains display profound and contrasting alterations in neuroendocrine, immunological and behavioral responses to stressors. Because of the established interactions between stressors, the neuroendocrine system, the immune system and pain processing pathways, we hypothesized that LEW and FIS rats would differ in their pain sensitivity. Pain sensitivity was assessed using several behavioral pain assays in untreated and carrageenan-inflamed LEW and FIS rats, and in the outbred Sprague-Dawley (SD) rat. The results showed that at baseline, FIS rats were the most sensitive to mechanical stimulation (the von Frey monofilament test) and the least sensitive to noxious heat pain (the Hargreaves radiant heat test). After intraplantar administration of carrageenan, LEW rats showed the least, and FIS rats showed the greatest, thermal hyperalgesia and mechanical allodynia/hyperalgesia. Hindlimb muscle grip force and tail-flick latencies did not differ across the three strains, either before or after carrageenan. These results demonstrate differences in basal and carrageenan-induced pain sensitivity in LEW, FIS and SD rats, which extend earlier findings that genetic factors modulate both basal and inflammatory pain. The results further demonstrate that basal pain sensitivity can be predictive of inflammatory pain sensitivity, with the direction of the effect dependent upon the pain measure.     

Maintenance of inhibitory interneurons and boutons in sensorimotor cortex between middle and old age in Fischer 344 X Brown Norway rats

Ultrastructurally identified inhibitory synapses in layer II of rat sensorimotor cortex decline between middle and old age [Poe, B.H., Linville, C., Brunso-Bechtold, J., 2001. Age-related decline of presumptive inhibitory synapses in the sensorimotor cortex as revealed by the physical disector. J. Comp. Neurol. 439, 65-72]. The current study investigated whether a loss or shrinkage of gamma-aminobutyric acid (GABA)ergic interneurons contribute to that decline. Coronal sections from middle-aged (15-17 months) and old (25-29 months) Fischer 344 X Brown Norway male rats were immunoreacted with antibodies to the GABA synthesizing enzyme glutamic acid decarboxylase (GAD); the calcium-binding protein parvalbumin (PV), or the neuronal marker NeuN. The number of GAD-immunoreactive (IR), PV-IR, and NeuN-IR cells were determined stereologically using the optical disector technique and the cross-sectional areas of GAD-IR cells were measured in layers II/III, IV, V and VI of sensorimotor cortex. Neither the number of GAD-IR or NeuN-IR cells, nor the size of GAD-IR cells, declined significantly between middle and old age. A modest decline in the PV-IR subset of inhibitory interneurons was observed, predominantly due to changes in layers V and VI. Stereological analysis of layer II/III GAD-IR boutons revealed a stability of immunocytochemically identified inhibitory terminals. Taken together, these results indicate a general maintenance of overall GABAergic neurons in sensorimotor cortex between middle and old age and the loss of ultrastructurally identified inhibitory synapses may be due to the decline of a subset of GABAergic terminals.     

An autoradiographic and immunocytochemical study of the neonatal rat pituitary gland.

Two-day-old female rats were injected with 5 nmole/kg of 6,7-3H-11 beta-methoxy-17-ethylestradiol (R 2858 = moxestrol) and killed one hour later. The animals were decapitated and, the pituitary glands were removed, mounted on tissue holders and frozen in liquified propane. The tissue was then processed for autoradiography according to the thaw-mount technique. At the end of the exposure time, prior to photographic development, some of the tissue was fixed in 10% formalin and then photographically developed for autoradiography. The fixed tissue was subsequently stained immunocytochemically using antibodies to luteinizing hormone or prolactin. Between 10 and 15% of the cells of the pars distalis concentrated the synthetic estrogen or its metabolite. The immunocytochemical procedure revealed that both LH-gonadotrophs and lactotrophs concentrated the steroid. These studies along with earlier studies suggest that the neonatal rat pituitary contains only a small portion of the adult complement of estrogen receptors and that these receptors are dispersed across a number of cell types.