Download felix may 2nd year neuroscience Evaluation of the effect of chronic

felix may
2nd year neuroscience
Evaluation of the effect of chronic treatment with methimazole on pituitary-thyroid axis
function in the rat
Aims
To analyse data from an experiment performed in rats.
Levels of thyroxine (T4) were assayed from the blood and thyroid glands, while thyrotropin-releasing
hormone levels were assayed from the hypothalamus and brainstem. There were two groups of animals,
one treated with methimazole for five days, and a control group.
Introduction
Assaying blood levels of hormones is a vital diagnostic tool used in clinics throughout the world. The
development of radioimmunoassays (RIA) for detecting pico-molar concentrations of hormones has
transformed the study of endocrinology. Today simple assay preperations are available for physicians to
assess a patient’s thyroxine levels, without access to lab facilities.
Thyroid hormones play a crucial role in homeostatic regulation, affecting the metabolic functioning of
every cell in an animal. Development and growth are modulated by thyroid hormones, as is the
sympathetic nervous system.
Dysfunction of the pituitary-thyroid axis causes and exacerbates many clinical conditions, including
Grave’s disease (an autoimmune hyperthyroidism) and Myxedema (a hypothyroid disorder, where
vasopressin and heart function are also affected, causing oedema). Thyroid function is also interwoven
with the endocrine control of the female reproductive system… indeed Grave’s disease is
symptomatically similar to normal thyroid changes that occur in pregnancy, making diagnosis tricky.
Two thyroid hormones, thyroxine and tri-iodothyronine (T4 and T3), are secreted from follicular cells in
the para thyroid glands found around the larynx. Thyroid hormone synthesis is stimulated by ciculating
thyroid-stimulating hormone (TSH) which is secreted from thyrotroph cells in the anterior pituitary. TSH
secretion is controlled by thyrotropin releasing hormone (TRH) released into the portal vessels from the
paraventricular nucleus of the hypothalamus. TRH secretion is increased by stimuli such as cold trauma
and high food intake. Both TSH and TRH secreting cells are inhibited by circulating thyroid hormones,
providing negative feedback to the axis.
T3 and T4 exert their effects through nuclear receptors, as well as some mitochondrial and membrane
targets. Within the cell T4 is converted to T3, thus T4 is a prohormone.
In the blood both hormones are 99.9% bound to thyroxine-binding globulin and prealbumin. Control of
thyroid secretions maintains the free hormone levels independent of protein binding, a thus free hormone
concentrations are a direct indication of thyroid status.
Thyroid homornes are lipid soluble and can cross cell membranes by diffusion.
Methimazole is an antithyroid drug that inhibits tyrosine peroxidase, and thus the organification of iodine
into the precursors monoiodotyrosine and diodotyrosine, preventing the biosynthesis of thyroid
hormones.
Methods
Rat TSH has a different amino acid sequence in its protein subunit to that in human TSH, and assays for
it are unavailable, so TRH was assayed as an indirect indication of pituitary-thyroid function.
Rats were treated with methimazole for 5 days at 40mg/kg or with a saline as controls. Over this period
the drug had time to equilibrate and the body to respond fully. Five animals were in each treatment
group, enough to provide reliable results, without excessive sacrifice.
Free plasma thyroxine (T 4) was assayed from mixed arterio-venous blood collected from the animals in
heparin coated tubes (to prevent blood clotting). T 4 is assayed rather than T3 as it has a longer half-life
and is present in higher concentrations.
Free T4 in the thyroid was assayed from a preperation of the thyroid gland. The tissue was dissected and
sonicated to lyse the cells and homogenise their contents. Methimazole was added to the veronal buffer to
prevent furthur production of thyroid hormones. The suspension was then centrifuged to remove cell
debris.
The Radio-immuno assay for free T 4 used a radiolabelled antibody and hormone bound to metallic beads.
Antibodies that bind to the free T 4 will remain in solution whereas those that bind the the beaded
hormone will be centrifuged out. The remaining activity gives a direct determination of the content of T 4.
Levels of TRH in the brain were also assayed… Both the hypothalamus and brainstem were prepared by
dissection and sonication in 90% methanol, then centrifuged. Extracting TRH from blood would yield
tiny quantities, as the hormone usually has to travel no futhur than down the portal veins to the pituitary
to exert it effects.
TRH levels were radioassayed using radiolabelled 125I-TRH, in the standard competition for antibody
binding with the unknown quantity of natural TRH.
Standard curves for the free T4 and TRH assays were drawn using data produced with known quantities
of radioactive and natural ligands. Using these curves the concentrations of hormones from the
experiment could be extrapolated.
Results
Blood free T4
mean SEM
methimazole
saline
180 ±56.4
600 ±81.2
Hypothalamus TRH
mean SEM
methimazole
saline
481 ±69.6
77.5 ±5.6
unpaired t-test
P
(P < 0.05)
0.0081 significant
unpaired t-test
P
(P < 0.05)
0.0044 significant
Discussion
From the results it can be seen that in rats who received methimazole treatment thyroxine levels were
significantly lower than in the saline treated group, both in blood and thyroid.
Upon t-testing the difference between groups for blood T 4 it was confirmed the the results were
significantly different.
Concentrations of T4 in the blood were proportionally lower than in the thyroid for both methimazole and
control groups. This is expected as the thyroid is the site of synthesis for T 4 and contains large stores of
the hormones.
TRH in the hypothalamus was significantly higher in the methizamole groups than the control rats. The
differences between groups for hypothalamic TRH concentrations are obviously great, and this was
confirmed by the t-test.
However TRH levels in the brainstem were largely unaffected by methimazole. Within the brainstem,
especially the dorsal raphe nuclei, TRH occurs as a neuropeptide. It is likely that TRH is synthesised by
neurons in the brainstem independent of the pituito-thyroid axis’ state.
Methimazole reduced the circulating concentrations of free T 4 (and presumably T3) as shown in the
experiment. The hypothalamus and the pituitary will respond to a fall in thyroid hormone concentrations
in the blood by releasing greater amounts of TRH and TSH, respectively. This experiment clearly
demonstarated the rise in TRH that would be predicted from the understanding of negative feedback in
the pituito-thyroid axis.
Treatment with anti-thyroid drugs aims to reduce the effects of thyroid hormones in the body, such as in
cases of hyperthyroidism. Methizamole acts to prevent the synthesis of tyrosine radicals, an essential step
in the formation of iodotyrosine, the precursor to thyroid hormones. Other potential targets for antithyroid
drugs include TRH and TSH receptors, although clinical medicines that act on those targets are yet to be
introduced.
Iodine is given in cases of hyperthyroidism as it (almost paradoxically) inhibit’s the secretion of thyroid
hormones and sends the thyroid glands into a semi-dormant state. This treatment is best for acute
hyperthyroidism as its effectiveness declines rapidly after a week or so.
Radioactive iodine is also given, but for more chronic depression of thyroid function. 131I is absorbed and
concentrated in the thyroid glands where it decomposes, after two months reaching effectively zero
activity. The beta radiation emitted during decomposition ionises the follicular cells, which is highly
cytotoxic. Thus the gland is directly restricted by culling a portion of its cells.
This experiment used a small number of animals to aquire results, and thus could be criticised for having
insignificant sample numbers. However the clear trends in results and coherence with the literature
suggest that the number of animals was enough for this purpose.
References:
Antepartal nursing management of Grave's disease - B. Karacic - Journal of Obstetric, Gynecologic, and Neonatal
Nursing, Vol 15, Issue 3 214-218
hypothalamic-pituitary-thyroid axis and the female reproductive system - Doufas AG, Mastorakos G. - Ann N Y Acad
Sci. 2000;900:65-76
Thyroxine treatment induces upregulation of renin-angiotensin-aldosterone system due to decreasing effective
plasma volume in patients with primary myxoedema - Cheol Whee Park, Young Shin Shin, Seog Ju Ahn, Suk Young
Kim, Eui Jin Choi, Yoon Sik Chang and Byung Kee Bang - Nephrol Dial Transplant (2001) 16: 1799-1806
Pharmacology 5th edition - Rang, Dale, Ritter, Moore
The Merck Manual of Diagnosis and Therapy, Chapter 8. Thyroid Disorders