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Biomedical subjects

H E Romeo

Publications and source records attributed to H E Romeo.

17 recordsLinked to original sources

Hypothalamic luteinizing hormone-releasing hormone content and serum luteinizing hormone levels in male rats during wallerian degeneration of sympathetic nerve terminals after superior cervical ganglionectomy.

The main hypothesis of this study was that sympathetic neurons located at the superior cervical ganglia (SCG) control luteinizing hormone (LH) releasing mechanisms by acting at a hypothalamic site. To test this, medial basal hypothalamus (MBH) luteinizing hormone-releasing hormone (LHRH) content and serum LH levels were measured in male rats subjected to superior cervical ganglionectomy (SCGx) or sham-operation 14 or 38 h earlier, at the time of degeneration of nerve endings post-SCGx. Significantly augmented MBH LHRH levels and decreased circulating LH were found in SCGx rats. In animals subjected to SCGx 14 h earlier and receiving a single injection of the alpha 1-adrenergic blocker prazosin, the beta-adrenergic blocker propranolol or a mixture of both drugs 45 min before sacrifice, only the injection of prazosin prevented the decrease of plasma LH levels. Neither treatment prevented the increase in MBH LHRH content. When prazosin was given every hour starting from the 10th to the 13th h after surgery, it was effective to prevent both the increase of MBH LHRH content and the decrease of serum LH found during sympathetic nerve degeneration. Similar repetitive injections of propranolol resulted in the greatest depression of serum LH, and in the greatest increase of MBH LHRH observed. Serum LH response to LHRH injection was similar in SCGx and sham-operated rats. The data indicate that SCG neurons exert, through inhibitory alpha 1-, and weaker, stimulatory beta-adrenoceptors, a significant influence on LHRH release at a supra-hypophysial site.

Animals

Compensatory parathyroid hypertrophy after hemiparathyroidectomy in rats feeding a low calcium diet.

The functional and anatomic compensatory response of the parathyroid gland was examined in hemiparathyroidectomized (HPTx) rats whose parathyroid hormone (PTH) secretion was stimulated by a low calcium diet. These responses were compared with those observed in the thyroid gland of hemithyroidectomized (HTx) rats. Rats kept on a low calcium diet for 10 days were subjected to HPTx, HTx, or sham operations. Throughout the experiment (up to 28 days after surgery), serum calcium levels of HPTx rats were lower than the basal, with delta values (mg/dl, mean +/- SEM) of -0.66 +/- 0.17 and -0.84 +/- 0.17, (P less than 0.05) 3 and 28 days after surgery, respectively. Serum PTH decreased significantly from 7 to 21 days after HPTx, reaching normality at day 28 after surgery. In HTx rats, serum thyroxine (T4) levels diminished significantly 7 days after surgery, and attained normality thereafter. The mitotic index (number of metaphases/1,000 cells) in parathyroid glands of colchicine-treated HPTx rats increased significantly in comparison to sham-operated controls, when examined 2 or 40 days after surgery. The mitotic index of thyroid follicular cells was significantly higher than that of their respective controls, 2 but not 40 days after HTx. These results indicate that after HPTx, a delayed compensatory response is found when the animals are kept under a low calcium diet. Parathyroid response is both delayed and of a minor degree compared to that found in the thyroid gland after HTx.

Adaptation, Physiological

Slower growth of tumours in sympathetically denervated murine skin.

In order to examine tumour growth in sympathetically denervated murine skin, two breast cancer tumour lines were employed, i.e. M3 tumours, of a relatively high local growth and low metastatic capacity, and MM3-LN tumours, that grew locally at a slower rate but disseminated early to the lung. Mice subjected to unilateral superior cervical ganglionectomy or sham-operation 2 weeks earlier were used. M3 or MM3-LN tumours were implanted in the ipsilateral ear to the surgical procedure. Tumour size was assessed every 2-6 days, starting from the 7th day after tumour implantation. Growth of M3 and MM3-LN tumours was significantly slowed by a previous sympathetic denervation of the skin territory. There were no significant differences in the number or size of pulmonary metastases at autopsy between mice subjected to ganglionectomy or to sham-operation. Ganglionectomy increased significantly ipsilateral submaxillary lymph node ornithine decarboxylase activity by 62% and decreased noradrenaline content to 8% of the innervated contralateral lymph node. The present results indicate a local inhibitory modulation of tumour growth by the sympathetic nervous system.

Adenocarcinoma

Evidence suggesting that the sympathetic nervous system mediates thyroidal depression in turpentine-induced nonthyroidal illness syndrome.

Acute superior cervical ganglionectomy (SCGx) induces in the rat a supraliminal release of neurotransmitter in the innervated tissues (i.e., thyroid gland). This temporary adrenergic hyperactivity is correlated with a significant depression of the thyroid economy resembling the nonthyroidal illness (NTI) syndrome in the rat, and suggest that the sympathetic nervous system may mediate thyroidal changes in NTI. In order to gain further insight into the thyroidal depression in the NTI syndrome, we studied the thyroidal norepinephrine (NE) turnover in turpentine oil (TURP)-induced NTI syndrome and the role of the cervical ganglia (SCG) in the development of NTI in the rat. TURP administration to sham operated rats induced a rapid and significant fall in plasma T4 and TSH levels, in the thyroidal response to exogenous TSH (TIU) and in the thyroidal NE content compared to controls (sham + saline) (T4: 3.1 +/- 0.3 vs. 5.1 +/- 0.6 micrograms/dl, respectively, mean +/- SE, p less than 0.02; TSH: 1.4 +/- 0.4 vs. 4.7 +/- 1.4 ng/ml, respectively, p less than 0.05; TIU: 92 +/- 14 vs. 201 +/- 20 cpm.microliter thyroid/cpm.mg plasma (T/P ratio), respectively, p less than 0.01; thyroidal NE: 680 +/- 20 vs. 761 +/- 29 pg/mg thyroid, respectively, p less than 0.05). The thyroidal turnover rate of NE, however, was significantly increased in TURP-injected rats compared to controls (122 +/- 13 vs. 86 +/- 10 pg/mg/h, respectively, p less than 0.05). TURP injection to chronic SCGx rats induced a similar fall in plasma TSH compared to controls (SCGx + saline) (1.3 +/- 0.2 vs. 4.3 +/- 1.1 ng/ml, respectively, p less than 0.02); plasma T4 and TIU, however, did not change significantly (T4: 3.4 +/- 0.4 vs. 3.7 +/- 0.3 micrograms/dl, respectively, NS; TIU: 172 +/- 8 vs. 226 +/- 27 T/P ratio, respectively, NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Anterograde nerve degeneration after superior cervical ganglionectomy coexists with a decrease in arginine vasopressin release in rats.

After bilateral superior cervical ganglionectomy (SCGx) in adult male rats, norepinephrine content of the pituitary neurointermediate lobe (NIL) decreased at 12-24 h after surgery to attain concentrations 40-60% of controls between 24 and 60 h after surgery. To assess arginine vasopressin (AVP) secretion during this time, plasma and NIL-AVP levels were measured by radioimmunoassay. In sham SCGx controls, plasma AVP increased about 2-fold within 6 h after surgery and decreased thereafter, to attain presurgical values by 60 h after surgery. In SCGx rats, a significant increase in plasma AVP concentration was observed at the 6th h after surgery, as compared to presurgical concentrations, with a decrease to values significantly lower than those of presurgical controls at 16-18 h after SCGx. As compared to sham-operated rats, significantly higher plasma AVP levels 6 h after surgery and significantly lower plasma AVP levels 16-24 h after surgery were found. NIL-AVP concentration in SCGx and sham-operated ras were significantly lower than presurgical levels at 6 h after surgery. SCGx rats had significantly higher amounts of AVP in NIL at 16-24 h after surgery. The changes in plasma and NIL-AVP levels found 6 or 16 h after SCGx or sham SCGx were unaffected by a prior pinealectomy. Two injections of the alpha 1-adrenoceptor blocker prazosin 45 and 90 min before sacrifice, alone or together with the beta-blocker propranolol, prevented the increase in plasma AVP found in SCGx rats 6 h after surgery, and the decrease in plasma AVP and the increase of NIL-AVP found 16 h after SCGx.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Increase in adrenocorticotropin release during wallerian degeneration of peripheral sympathetic neurons after superior cervical ganglionectomy of rats.

The changes in adrenocorticotropin (ACTH) release before, during and after sympathetic nerve degeneration following superior cervical ganglionectomy (SCGx) were examined in male rats. A 12-fold increase of circulating ACTH was found in both SCGx and sham-operated rats 6 h after surgery. In sham-operated rats, plasma ACTH decreased by about half 16-22 h after surgery, whereas in SCGx rats it remained at a high concentration from 16 to 54 h after surgery, attaining basal values by 120 h post-SCGx. In SCGx rats, MBH corticotropin-releasing hormone (CRH) content decreased significantly from 16 to 54 h after surgery, while in controls it remained unmodified. Significantly smaller arginine vasopressin (AVP) contents were found in MBH of SCGx rats as compared to sham-operated controls, 16-54 h after surgery. In rats exposed to ether or immobilization stress 22 h after SCGx, plasma ACTH levels were significantly higher than in controls; however, since unstressed ACTH levels were about twice as high in SCGx rats, the percent increase of ACTH was smaller in the SCGx group. A decreased response of plasma ACTH to ether or immobilization stress was found in rats 7 days after SCGx. In rats subjected to a simultaneous adrenalectomy (Adx) and SCGx or sham-SCGx, plasma ACTH levels increased to a similar extent in both groups. ACTH increase after Adx was accompanied by decreases in MBH CRH, and absence of significant changes in MBH AVP contents. Rats subjected to pinealectomy (Px) or sham-Px 1 week earlier and killed 22 h earlier exhibited similar responses in plasma ACTH and MBH CRH to SCGx regardless of pineal intactness.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone

Estrous cycle delay and inhibition of gonadotropin and prolactin release during sympathetic nerve degeneration after superior cervical ganglionectomy of rats.

The changes in the activity of the pituitary-gonadal axis during sympathetic nerve degeneration after superior cervical ganglionectomy (SCGx) were examined in female rats. In a first experiment SCGx performed at 24.00 h of diestrus II, 17 h in advance to the critical period for gonadotropin and prolactin (PRL) release, caused a delay of 1 day or more in estrous cyclicity, while SCGx at 24.00 h on estrus did not modify the estrous cycle. In a second experiment ovariectomized rats injected subcutaneously with estradiol-progesterone or vehicle were subjected to SCGx 17 h before the expected maxima in luteinizing hormone (LH), follicle-stimulating hormone (FSH), and PRL serum levels. A significant decrease of serum gonadotropin and PRL concentration was found in SCGx, steroid-treated rats. In a third experiment groups of rats injected with estradiol-progesterone and killed at 15.00, 16.00, 17.00, or 18.00 h at or around the time of maximal FSH, LH, and PRL release were used. SCGx performed 17 h before killing caused a generally depressive effect of LH, FSH, and PRL release. In a fourth experiment ovariectomized rats were subjected to pinealectomy and, 1 week later, to estradiol-progesterone treatment and SCGx as in experiment 2. Pinealectomy did not modify the depression of steroid-induced LH, FSH, and PRL release found during wallerian degeneration of sympathetic nerves after SCGx. In a fifth experiment the effect of alpha 1- and/or beta-adrenoceptor blockade on SCGx-induced inhibition of hormone release was assessed in estradiol-progesterone treated, spayed rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

24 hour changes in catecholamine content of rat thyroid and submaxillary glands.

The occurrence of diurnal rhythmicity in tissue norepinephrine (NE), epinephrine (E) and dopamine (DA) levels of rat thyroid and submaxillary glands was assessed in animals killed at eight time intervals during a 24 hour period. In both tissues significant peak values of NE content were found at the third hour of dark onset (at 23:00 h). Additionally a minimum of NE content of thyroid gland was apparent in rats killed at the fifth hour of photophase (at 11:00 h). As in the case of NE, peak values of E content were found during the scotophase in both tissues examined. In contrast, DA levels exhibited no diurnal rhythmicity in the rat thyroid gland, and an early rise at the beginning of the photophase in the submaxillary glands. These results support an activation of the sympathetic nervous system innervating the thyroid and submaxillary glands at the beginning of the scotophase. Tissue DA levels do not resemble the activity of peripheral sympathetic nerves in the tissues examined.

Animals

Benzodiazepine binding sites in rat interscapular brown adipose tissue: effect of cold environment, denervation and endocrine ablations.

3H-Flunitrazepam (FNZP) binding was examined in a crude membrane fraction obtained from rat interscapular brown adipose tissue (IBAT). A single population of binding sites was apparent with dissociation constant (KD) = 0.47 +/- 0.04 microM and maximal number of binding sites (Bmax) = 31 +/- 5 pmol.mg prot-1. From the activity of several benzodiazepine (BZP) analogs to compete for the binding, the peripheral nature of FNZP binding was tentatively established. Similar BZP binding sites were detectable in isolated IBAT mitochondria. Exposure of rats to 4 degrees C for 15 days decreased Bmax significantly without affecting KD. Cold-induced decrease in Bmax of BZP binding was prevented by surgical IBAT denervation. Denervation prevented or impaired the increased activity of the mitochondrial markers succinate dehydrogenase and malate dehydrogenase in IBAT of cold-exposed rats, but did not affect monoamine oxidase activity. Hypophysectomy of rats decreased significantly both KD and Bmax of IBAT BZP binding. Thyroidectomy, adrenalectomy or ovariectomy did not affect IBAT BZP binding parameters. The BZP analogs diazepam, clonazepan and Ro 5-4864 decreased significantly guanosine 5'-diphosphate binding (GDP) in IBAT mitochondria while co-incubation of Ro 5-4964 or clonazepam with the peripheral type BZP antagonist PK 11195 did not modify BZP activity on GDP binding. Our results indicate that BZP binding in rat IBAT may belong to the peripheral type, is decreased by a cold environment through activation of peripheral sympathetic nerves and is affected by hypophysectomy. BZP and GDP binding in IBAT mitochondria seem not to be functionally related.

Adipose Tissue

Effect of superior cervical ganglionectomy on insulin release by murine pancreas slices.

The effect of superior cervical ganglionectomy (SCGx) on basal and glucose-stimulated insulin release in vitro was examined in pancreas slices of BALB/c mice subjected to surgery 14-96 h earlier. Fourteen or 20 h after SCGx a significant increase of insulin response to 11 mM glucose was detectable, while 96 h after SCGx a depression in response was found. Perifused pancreas slices obtained from mice subjected to SCGx 7 days earlier showed a decreased in vitro insulin response to glucose during both phases of insulin secretion. In sham-operated mice, injection of the beta-adrenoceptor blocker propranolol or of the cholinergic muscarinic antagonist atropine decreased basal and glucose-stimulated insulin release, while the alpha-adrenoceptor blocker phenoxybenzamine did not affect it significantly. In mice subjected to SCGx 14 h earlier, propranolol treatment decreased basal insulin release and impaired the release elicited by glucose to values similar to those found in controls, phenoxybenzamine injection increased the basal and amplified the enhanced glucose-stimulated insulin release, and atropine injection, although unable to affect basal insulin release, impaired partially the amplification of response detectable after surgery. Our results support the existence of significant effects of SCG neurons on insulin release in mice.

Animals

Effect of inferior laryngeal nerve section on thyroid function in rats.

To examine the role of thyroid parasympathetic innervation in organ's function, rats subjected to inferior laryngeal nerve (ILN) section were employed. This procedure decreased thyroid [3H]choline uptake by about half. Bilaterally ILN-sectioned rats treated with methylmercaptoimidazole for 4 days exhibited a significant impairment of the methylmercaptoimidazole-induced goitrogenic response. Unilateral ILN section resulted in further atrophy of the ipsilateral thyroid lobe in hypophysectomized rats. One week after ILN section a significant decrease of serum T4 and an increase of serum TSH were observed. Bilateral ILN section generally decreased circulating T4 for up to 28 days after surgery, while a unilateral ILN section caused a transient T4 decrease for 1 week after surgery. Compensatory thyroid growth in rats subjected to unilateral thyroidectomy (hemi Tx) and ILN section performed ipsilaterally to the remaining lobe, was significantly smaller than that of rats subjected to hemi Tx alone. Hemi Tx depressed serum T4 and increased serum TSH levels significantly. These hormonal changes were prevented by unilateral superior cervical ganglionectomy (SCGx), but were unaffected by ILN section. The combination of SCGx and ILN section negated the facilitating effect of SCGx on thyroid secretion and impaired the increase in compensatory thyroid growth brought about by thyroid sympathetic denervation. Thyroid mitotic index studies in hemi Tx rats receiving ILN section, SCGx, or a combination of both indicated that the increase in the number of follicular mitosis caused by hemi Tx was significantly impaired by ILN section and was significantly increased by SCGx. SCGx potentiation of thyroid follicular mitotic activity was partially prevented by concomitant ILN section. These results support a significant role of thyroid sympathetic and parasympathetic innervation in the control of organ's growth and secretory activity.

Animals

Autonomic nervous system regulation of murine immune responses as assessed by local surgical sympathetic and parasympathetic denervation.

The effect of local sympathetic denervation on immune responses of submaxillary lymph nodes (SmLN) of mice was examined in animals subjected to unilateral superior cervical ganglionectomy (SCGx). Norepinephrine (NE) content in ipsilateral SmLN decreased by 90% 7-20 days after SCGx, while bilateral SCGx resulted in a 91% decrease of SmLN NE content. SmLN of mice subjected to unilateral SCGx exhibited greater plaque-forming cell (PFC) response than the innervated contralateral SmLN, when challenged i.d. or i.p. with sheep red blood cells (SRBC) 10-20 days after surgery. In mice challenged with SRBC at an early phase of sympathetic nerve paralysis (i.e., 2 h after SCGx), PFC response was already increased in the ipsilateral SmLN whereas during the anterograde degeneration of nerve endings (6-24 h after SCGx) an impending depression of PFC number was observed. Contact hypersensitivity and allogeneic delayed-type reactions were also enhanced in the ear ipsilateral to SCGx. Primed SmLN cells of nodes located ipsilaterally to SCGx, when injected to (BALB/c x C57BL/6) F1, resulted in significantly higher spleen index than similarly treated, contralateral SmLN. In contrast, mitogenic stimulation under various experimental protocols failed to reveal significant differences between proliferation of cell populations obtained from ipsilateral and contralateral SmLN of unilaterally SCGx mice. SmLN of mice parasympathetically decentralized by unilateral sections of the chorda tympani-lingual nerve trunk showed lower PFC response than the innervated contralateral SmLN, when challenged with SRBC 8-28 days after surgery. These results suggest a regulatory function of the autonomic nervous system in immune reaction.

Animals

Origins of the sympathetic projections to rat thyroid and parathyroid glands.

The present study aimed to characterize the localization and pathways of sympathetic neurons innervating the thyroid and parathyroid glands. In rats subjected to unilateral superior cervical ganglionectomy or unilateral decentralization of the superior cervical ganglion 7 days earlier, ipsilateral depression of thyroid norepinephrine (NE) and epinephrine content to 6-16% of the contralateral intact lobe was observed. In both groups of animals neuronal [3H]NE uptake by the ipsilateral thyroid lobe was suppressed. In unilaterally decentralized rats pineal catecholamine levels remained within normal values whereas in unilaterally ganglionectomized rats a 74% decrease of pineal NE and E content was found. Unilateral section of the external carotid nerve abolished, as did unilateral superior cervical ganglionectomy, neuronal [3H]NE uptake in the ipsilateral thyroid lobe. In contrast external carotid nerve section did not modify the neuronal uptake of [3H]NE in the pineal gland. Either unilateral superior cervical ganglionectomy or external carotid nerve section resulted in similar involution of ipsilateral thyroid lobes of hypophysectomized rats. These results indicate that postganglionic sympathetic perikarya innervating the thyroid-parathyroid territory are located in the middle and/or inferior cervical ganglia and send their axons through the SCG and the external carotid nerve to these glands.

Animals

Early inhibition and changes in diurnal rhythmicity of the pituitary-thyroid axis after superior cervical ganglionectomy of rats.

The effect of superior cervical ganglionectomy (SCGx) on the pituitary-thyroid axis was examined in rats. SCGx decreased serum thyrotropin (TSH) and thyroxine (T4) levels for up to 4 days after surgery, during and immediately after completion of anterograde degeneration of regional sympathetic terminals. At later times TSH levels in control and SCGx rats did not differ, but a significant increase of serum T4 was found two weeks after SCGx. A diurnal rhythm in serum TSH and T4 levels with maxima at 11.00 h (TSH) and at 14.00 and 22.00 h (T4) was found in sham-operated rats 3 days after surgery. At this time SCGx evoked a general depression of TSH levels as well as a shift of 3 h in their maximum. A similar shift of the afternoon peak and abolition of the nocturnal peak in serum T4 were detectable in SCGx rats. In SCGx animals examined during anterograde nerve degeneration, i.e. 14 h after surgery, injection of the alpha 1-adrenoceptor blocker phenoxybenzamine negated denervation-induced changes of TSH and counteracted partially T4 effects. The beta-adrenergic blocker propranolol did not modify serum TSH levels in SCGx rats but further decreased serum T4 concentration. Treatment with both drugs simultaneously did no affect TSH release compared to SCGx, phenoxybenzamine-treated rats but effectively decreased serum T4. These results further support the involvement of superior cervical ganglion neurons in the control of thyroid function.

Animals

Changes in gamma-aminobutyric acid high affinity binding to cerebral cortex membranes after pinealectomy or melatonin administration to rats.

In order to assess the effect of pinealectomy (Px) on the diurnal rhythmicity of gamma-aminobutyric acid (GABA) high affinity binding to cerebral cortex membranes, groups of intact, Px or sham Px rats (subjected to surgery 15 days earlier) were killed at six different time intervals during the 24-hour cycle. GABA binding was estimated by Scatchard analysis of 3H-GABA binding to cerebral cortex membranes prepared from individual brains; only one type of binding site with dissociation constant (KD) about 20-50 nM and site number (Bmax) about 200-500 fmol/mg protein was apparent in the assay conditions employed. In intact and sham Px rats Bmax attained minimal values at night and increased during daylight. Px increased generally Bmax and disrupted its normal diurnal rhythmicity, a peak in Bmax being observed at midnight. A significant decrease of GABA high affinity binding affinity was detected at morning hours in intact rats and at late scotophase and morning hours in Px and sham Px rats. Bmax of GABA high affinity binding in Px rats attained maximal values by 5-10 days after surgery and decreased somewhat 5 days later. Sham Px rats exhibited a transient increase in Bmax up to 10 days after surgery, returning to normal values by the 15th day. Superior cervical ganglionectomy increased binding affinity up to 15 days after surgery without affecting Bmax. The minimal melatonin effective dose to counteract Px-induced increase of GABA high affinity binding was 25 micrograms/kg body weight when given 3 h before sacrifice. Melatonin activity on GABA binding did not depend upon a direct effect on the binding sites, as shown in vitro. These results suggest a link between pineal function, melatonin secretion and GABA receptor activity in rats.

Animals

Role of the sympathetic nervous system in the control of thyroid compensatory growth of normal and hypophysectomized rats.

Compensatory thyroid growth in rats subjected to unilateral thyroidectomy (Tx) and superior cervical ganglionectomy (SCGx) performed ipsilaterally to the remaining lobe was about 2-fold that of rats subjected hemiTx alone, when assessed 7-15 days after surgery. HemiTx depressed serum T4 levels by 33% and increased serum TSH by 80% in control rats. Unilateral SCGx, which did not affect circulating T4 or TSH by itself, prevented hemiTx-induced changes of hormone levels. After hypophysectomy (Hpx) thyroid involution ensued. The subsequent hemiTx of Hpx rats did not affect Hpx-induced thyroid regression 7 days later, but abolished it 15 days later. At this time a factorial analysis of variance revealed lack of significant interaction between Hpx and hemiTx thus suggesting that a similar thyroid growth ensues after hemiTx regardless of pituitary intactness. However, both serum T4 and TSH levels were very low in Hpx rats regardless of whether one or two thyroid lobes were present indicating that thyroid growth in the absence of pituitary was not accompanied by increased secretory activity. SCGx performed ipsilaterally to the remaining lobe blunted the growth of the lobe in Hpx hemiTx rats. Moreover, the denervated thyroid lobe regressed in Hpx rats to values significantly lower than those of Hpx rats having intact sympathetic nerves both in the presence or in the absence of a contralateral thyroid lobe. SCGx did not modify the very low serum TSH and T4 levels of Hpx rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals