Vagosympathetic imbalance induced thyroiditis following subarachnoid hemorrhage: a preliminary study

1Department of Pediatric Surgery, Medical Faculty of Ataturk University, Erzurum, Turkey 2Department of General Surgery, Medical Faculty of Ataturk University, Erzurum, Turkey 3Department of Anesthesiology and Reanimation, Medical Faculty of Ataturk University, Erzurum, Turkey 4Department of Pathology, Medical Faculty of Ataturk University, Erzurum, Turkey 5Department of Neurosurgery, Medical Faculty of Ataturk University, Erzurum, Turkey


Introduction
Thyroid gland is innervated by vagal parasympathetic, cervical chain sympathetic and cervical somatosensitive fibers. Parasympathetic insufficiency or sympathetic overactivity may cause autoimmune thyroid disease.
Its most frequently detected form is Hashimoto thyroiditis, more frequently diagnosed in women 1 with characteristically increased anti-thyroglobulin antibodies, 2 elevated thyroid-stimulating hormone, and low thyroxine levels. 3 Lymphocytic/eosinophilic infiltration, follicular atrophy, 4 fibrosis, and scar fibrosis are commonly found in Hashimato's thyroiditis. 5 The suprachiasmatic nucleus (via TSH release), 6 superior cervical/intrathyroidal ganglia of vagal nerves, trigeminal nerve, and cervical dorsal root ganglia regulate thyroid gland functions. 7 Cervical sympathetic fibers decrease vascular conductance 8 and parasympathetic system increases secretory activity. 9 Based on the studies of Sundler et al, 7 we postulated that vagal insufficiency triggering relative adrenergic hyperactivity could be an important underestimated cause of autoimmune thyroiditis following subarachnoid hemorrhage (SAH). This has not yet been reported in the literature.

Methods
This study was conducted on 26 rabbits chosen from former SAH experiments. All rabbits were male hybrid at the age of 3±0.4 years and weighed 4.2±0.3 kg. Five healthy rabbits were included as controls (group-I), five were included in the sham group (group-II) and twelve rabbits diagnosed with severe hypothyroidism in former experiments were included in the study group (group-III).
Among the study group, six rabbits were chosen from the group with severe hypophyseal ischemia (GIII-A) and six were picked from the vagal ischemia group (GIII-B). All animals were followed in special metal cages at normal living room standards.
All animals were examined as four groups: Five rabbits were selected for the control group (group I, n=5) without surgical application. Five rabbits were used as sham group (group-II, n=5) chosen from animals with one mL of serum saline injection in their cisterna magna. Our experimental procedure as follow; to reduce pain and mortality, 0.2 mL/kg of isoflurane with ketamine HCL 150 mg/1.5 mL, xylazine HCL 30 mg/1.5 mL, and distilled water 1 mL was used. After bringing the head to a hyperflexed position to identify the foramen magnum, a 22-gauge needle was inserted in the cisterna magna and cerebrospinal fluid was aspirated. Half a milliliter of autologous blood was injected over about one minute to induce a SAH condition (G III, n=12). Surgical techniques related to autologous blood injection into cisterna magna were represented on a 3-D computerized tomography of the rabbit (Figure 1). They were monitored for vital signs with ten minutes periods two times a week and hormone levels were evaluated during the experiment. The thyroid glands, vagal nerves and their ganglia, together with other brainstem nerves, were kindly removed from all rabbits.
The brains, vagal networks, cervical sympathetic chains, and thyroid tissues were washed with a graded alcohol series and embedded in liquid paraffin.

Histopathological procedures
The thyroid glands, superior cervical ganglia, and For the analysis of vagal nuclei lesions, brain specimens were sectioned parallel to the long axis of these nuclei to examine both brainstems at that level and were stained with H&E and TUNEL dyes. To estimate the neuron density of the vagal nucleus, all of the vagal nerves together with their extensions, were embedded in paraffin blocks.
Stereological method was used to analyze vagal neuron density, as in our previous studies. 10

Stereological analysis
Stereological methods were used to provide good tools to estimate the total volume of thyroid follicles, sympathetic ganglia, and vagal web elements. The first sampled sections pair was selected randomly from a starting point within the first 30-section interval. Thereafter, every 30 th section and its neighbor were sampled. The follicular sections sampling fractions of thyroid gland (f1) were therefore, f1=1/30. Section pairs not containing the thyroids cells and vagal neuron nuclei were discarded. This sampling fraction yielded on average 10 to 11 section pairs. Area of sampling fraction (f2) was 1/1. We preferred to use the physical dissector method to evaluate the number of thyroid follicles, sympathetic ganglia neurons, and vagal complex particles to minimalize bias, as in our former studies. 11,12 Numerical results of all specimens were estimated as in our previous studies. Vagal circuitry neuron densities were calculated with the method described by Aydin et al 13 and the thyroid gland was examined as described by Aydin et al. 11 Vasospasm index calculation method was used to determine the severity of the thyroidal artery spasm, as described by Ozoner et al. 10 The data were analyzed using a commercially available statistics software package (SPSS ® for Windows v. 12.0, Chicago, USA). The data were analyzed with the Kruskal-Wallis and Mann-Whitney U tests. Differences were considered significant at P < 0.05.

Anatomical and histopathological results
Macroscopic appearance of a SAH-created brain, histopathological appearance of SAH just over the hypophyseal stalk, internal carotid arteries, and spastic cortical branch of hypophyseal artery are seen in a rabbit with SAH in Figure 2.   from intrathyroidal lymphatic tissues to thyroid gland with follicular destruction. Figure 9 shows lessened/atrophic thyroid follicles in severe thyroidal arterial vasospasm, immunologic staining of the same thyroid gland, and the atrophic/fibrotic thyroid gland.

Numerical results
The mean neuron density of both superior cervical ganglia was estimated as 8230±983/mm 3 in animals with severe thyroiditis, 7496±787/mm 3 in the sham group and 6416±510/mm 3 in animals with normal thyroid glands.

Discussion
Autoimmune thyroiditis is the most frequent autoimmune

Autonomic innervation of thyroid gland
Generally, the thyroid gland is controlled by the suprachiasmatic nucleus via their TSH release, 6 superior cervical ganglia, intrathyroidal ganglia of vagal nerves, trigeminal ganglia, cervical dorsal root ganglia which their fibers project to the thyroid gland and control of thyroid activity. 7 The thyroid gland and blood vessels have double innervation arising from the sympathetic and parasympathetic fibers. 16 They are located in the periphery of arterioles under the fibrous capsule and around secretory vesicles. 17 Cervical sympathetic trunk stimulation decreases vascular conductance. 8

Central innervation
The thyroid gland is generally controlled by the suprachiasmatic nucleus via their TSH release. 6

Parasympathetic innervation
The

Sympathetic innervation
Cervical sympathetic ganglia send their axons around external carotid arteries to the thyroid gland. 19 Sympathetic innervation of the thyroid gland is provided by superior cervical ganglion, 20 which contributes to gland enlargement and may modulate tissue/TSH interactions. 21 The sympathoadrenal system interacts with thyroid

Effect of sympathicovagal network imbalance on thyroid gland
Hypothalamo-hypophyseal axis injury causes TSH insufficiency induced thyroid dysfunctions, 6 extirpation of the nodose ganglion, and decrease of parasympathetic effect on thyroid gland. 24 The unilateral superior cervical ganglion section leads to a decrease in the size of the thyroid gland. Postganglionic nerve transection causes various histomorphological abnormalities on related glands. 23 The unilateral superior cervical ganglion section leads to a decrease in the size of the thyroid gland. Acutely superior cervical ganglionectomy has a significant role on the depression of the thyroid economy. 25

Evaluation of our results in the light of current literature
Although hypothalamo-hypophyseal axis pathologies have been the scapegoat following SAH, this study has revealed that various neurophysiological mechanisms may be responsible additionally, because it is shown that hypothalamo-hypophyseal axis injuries do not cause the expected biochemical/histopathological deterioration.
This astonishing finding led us toward examining the peripheral autonomic neural network that innervates the thyroid.
Our histopathological examinations showed that ischemic degeneration in vagal nuclei and nodose ganglia could result in thyroid gland dysfunction due to parasympathetic denervation deficiency. In the meantime, a large number of neurons included cervical sympathetic ganglia or vagal network ischemia-induced indirect sympathetic overactivity could be responsible for excessive catecholamine release induced thyrotoxicosis likely described by previous authors. 22 In conclusion, we could say with a great insight that the damage of the peripheral autonomic network innervating the thyroid gland can be as responsible or even more than the hypothalamic-pituitary axis in the pathology of autoimmune thyroiditis.

Rationale of the study
Vagosympathetic circuitry plays an essential role on thyroid anatomy, histology and physiology with parasympathetic metabolism and sympathetic catabolism functions. Vagal insufficiency causes decreased blood flow and immunological disorders of thyroid. Sympathetic discharges have catabolic and inflammatory effects. SAH models dangerously affect lower cranial nerves, such as the vagal nerves. Our macroscopic and microscopic examinations revealed that blood clots and inflammation in needle insertion sites have compressive effects on vagal nerves. Histopathological examinations have also shown pia-arachnoid adhesion, inflammation, and spasm in vagal nerve root supplying arteries and vagal network result in ischemia. However, SAH rarely results in sympathetic ischemia secondary to external carotid artery spasm. 26 For that reason, SAH induced vagal ischemia and relatively augmented sympathetic overactivity may be responsible for thyroiditis. As seen in Figure 8, lymphoid cells infiltrating the thyroid follicles is our most prominent histopathological evidence.

Limitations
Stronger data would be available if radiological analyzes were also performed.

Future insight
Although sympathetic hyperactivity has been accused of the etiology of Hashimoto's thyroiditis within the last century, vagal insufficiency induced sympathetic hyperactivity has not been mentioned, and even regrets of that reality initiates the dark age in medical science.
We are expecting future researchers to consider the mentioned histopathological mechanism and to conduct critical studies in the future. Vagal stimulation may be a new treatment method for autoimmune thyroid diseases.

Conclusion
Vagal network ischemia based relative sympathetic overactivity may cause thyroid tissue destruction.
Destructed follicular cell remnants are considered foreign antigenic materials, and the thyroidal lymphocytic cells are stimulated to exterminate them. As a result, thyroid tissue enlarges with activated intrathyroidal lymphatic nodes, which causes the nodular thyroid gland appearance. In the progression of that neuropathological war, the thyroid gland becomes atrophied and sclerosed like as Hashimoto's thyroiditis. We concluded that sympathovagal imbalance following SAH might be considered the unpublished neuropathological mechanism of Hashimoto thyroiditis,