- Department of Neurosurgery, Sisli Research and Education Hospital, Istanbul, Turkey
- Department of Pathology, Ataturk University Medical School, Erzurum, Turkey
- Department of Neurosurgery, Ataturk University Medical School, Erzurum, Turkey
- Department of Neurosurgery, Rize University Medical School, Rize, Turkey
Correspondence Address:
Mehmet Dumlu Aydin
Department of Neurosurgery, Sisli Research and Education Hospital, Istanbul, Turkey
DOI:10.4103/2152-7806.82084
Copyright: © 2011 Yilmaz A. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.How to cite this article: Yilmaz A, Cemal Gündoğdu, Aydin MD, Musluman M, Kanat A, Aydin Y. Trigeminal ganglion neuron density and regulation of anterior choroid artery vasospasm: In a rabbit model of subarachnoid hemorrhage. Surg Neurol Int 15-Jun-2011;2:77
How to cite this URL: Yilmaz A, Cemal Gündoğdu, Aydin MD, Musluman M, Kanat A, Aydin Y. Trigeminal ganglion neuron density and regulation of anterior choroid artery vasospasm: In a rabbit model of subarachnoid hemorrhage. Surg Neurol Int 15-Jun-2011;2:77. Available from: http://sni.wpengine.com/surgicalint_articles/trigeminal-ganglion-neuron-density-and-regulation-of-anterior-choroid-artery-vasospasm-in-a-rabbit-model-of-subarachnoid-hemorrhage/
Abstract
Background:Subarachnoid hemorrhage (SAH) is associated with severe vasospasm caused by a variety of neurochemical mechanisms. The anterior choroid arteries (AChAs) are innervated by vasodilated fibers of the trigeminal ganglion (TGG). The goal of this study was to determine whether there is a relationship between the neuron density of the TGG and the severity of AChAs vasospasm with SAH.
Methods:Thirty-two rabbits were used for the study; eight served as the baseline control group, seven as a SHAM group, with injections of 1 cc of isotonic saline solution, and 17 rabbits were included in the experimental SAH group, with injection of homologous blood into the cisterna magna. After 10 days, the histopathology of the AChAs and TGGs were examined. The AChAs vasospasm index (VSI) of the external/internal diameter and the neuron density of the ophthalmic root of the TGGs were evaluated stereologically. The AChAs VSI was preferred - a measure of the degree of vasospasm. As the VSI increased, the degree of arterial vasospasm increased. The results were statistically analyzed.
Results:The mean AChAs VSI was significantly higher and the mean neuronal density of the ophthalmic root of the TGG was significantly lower in the group with severe vasospasm associated with SAH compared to the controls, SHAM, and the group with mild vasospasm associated with SAH (P
Conclusions:The trigeminal ganglion neuron density may be an important factor in the regulation of AChAs diameter and cerebral blood flow. Low neuron density of the ophthalmic root of the TGG may play a role in the pathogenesis of AChAs vasospasm associated with SAH.
Keywords: Anterior choroid artery, neuron density, subarachnoid hemorrhage, trigeminal ganglion, vasospasm, vasospasm index
INTRODUCTION
Cerebral vasospasm is a serious complication of subarachnoid hemorrhage (SAH); delayed narrowing of the large capacity arteries and cerebral vasospasm are associated with significant morbidity and mortality following a SAH.[
The cerebral arteries are innervated by several systems that contribute to the autonomic control of cerebral blood flow. Parasympathetic fibers affect vasodilation and sympathetic fibers cause vasospasm of the cerebral arteries.[
Neuro-humoral mechanisms have been suggested as important factors associated with the cerebral artery vasospasm after a subarachnoid hemorrhage.[
MATERIALS AND METHODS
Thirty-two anesthetized, adult male New Zealand rabbits were used for this study. The Ethics Committee of Atatürk University, Medical Faculty, approved the animal protocols. Animal care and experimental protocols were conducted according to the guidelines set forth by the same ethics committee. The animals were randomly divided into three groups: SAH (n=17), isotonic saline solution (SHAM; n=7), and control (n=8) groups. A balanced, injectable anesthetic was used to reduce pain and mortality. Anesthesia was induced with isoflurane, given by a facemask, and 0.2 mL/kg of the anesthetic combination of Ketamine HCL, 150 mg/1.5 mL; Xylazine HCL, 30 mg/1.5 mL; and distilled water, 1 mL, was subcutaneously injected prior to the surgery. All animals were monitored for changes in the electrocardiogram, respiration patterns, and blood oxygen concentration during the experiment. All parameters were recorded by a camera and analyzed by physicians that did not know to which experimental group the individual animals belonged. During the procedure, a dose of 0.1 mL/kg of the anesthetic combination was used when required. In 17 (n=17) of the animals, autologous blood (0.5 mL) was taken from the auricular artery. While the head of the animal was held in a hyperflexed position, the posterior notch of the foramen magnum was identified, and the cisterna magna was entered; CSF was then aspirated. When the identification of the cisterna magna was confirmed, blood was injected using a 22-gauge needle, over about 1 minute, in the SAH group; 1 mL of isotonic saline solution was injected in the same way in the seven (n=7) animals of the SHAM group. The remaining eight (n=8) animals not subjected to this procedure were considered the control group. The animals were followed for 10 days without any medical treatment and then killed. The time was selected based on relief of the vasospasm. The number of ganglion neurons is thought to be associated with vasospasm. The effects of SAH on vasospasm were studied by the removal of all AChAs and trigeminal ganglia, bilaterally, for histological examination. Specimens were stored in a 10% formalin solution for 7 days, after which 5 μm tissue sections were cut and stained with hematoxylin and eosin.
To estimate the neuron density of the ophthalmic division of the trigeminal nerve, all trigeminal roots, together with their ganglions, were extracted bilaterally. The specimens were then horizontally embedded in paraffin blocks to evaluate all roots during the histopathological examination. The physical dissector method[
Figure 1
(a, b) Stereologic cell counts of the ophthalmic division of the TGG in a rabbit. Use of the physical dissector method where the micrographs of the same fields of view (a, b) were taken from two parallel, adjacent thin sections separated by 5 μm. The numerical density of the neurons was calculated as NvGN= ∑Q – N/t × A. In this application, the nucleoli marked as ‘2,3,4,8’ are dissector particles in A. Section B shows them as they disappeared. The nucleoli marked as ‘1,5,6,7’ do not represent dissector particles in A. Section B shows ‘1 and 8’ as they disappeared (H and E, 100, LM)
The AChAs were obtained from the coronal brain sections at the level of the AChAs entering into the lateral ventricles. They were also stained with H&E. For the calculation of the vasospasm index of the AChAs, all AChAs were accepted as a cylinder, in view of their morphological characteristics; simple geometric formulas were used to estimate their wall ring values. As a measure of the degree of vasospasm, the use of the AChAs vasospasm index was preferred to use of only measurements of the lumen radius; it can be readily performed, is intuitively simple, more reliable, free from assumptions about vessel diameter of various segments and is unaffected by overestimation errors of the radius of the AChAs. The AChAs of all animals were cut 20 segments away from the point where the internal carotid arteries entered the choroid plexus. Then, 20 histopathology sections, 5 μm apart, were obtained, using a microtome, for each designation, and are represented by the lines 1, 2, 3,… and 20. The average of 10 diameters, of 10 cross-sectional areas, was recorded as the mean diameter. A single line in the figures represents one of them. The mean external and internal (luminal) diameters of each section was measured; the external radius is represented by R1 and the internal radius is represented by r1. The mean external radius of the anterior choroid arteries was calculated as R1 = R1 + R2 + R3 +….R20/20; the lumen radius was calculated as r1 = r1 + r2 + r3 +….r20/20 [
In summary, VI= (R12 – r12)/r12. In addition, the vasospasm index of AChAs in the rabbits with SAH was calculated as the same manner; VI= (R22 – r22)/r22 . As the VSI increases, the degree of arterial vasospasm also increases. Vasospasm was defined as mild with a reduction of 25% of the normal diameter of any arterial segment of AChA and as severe vasospasm with a decrease of 40%.[
The differences between the VSI of AChAs and neuron density of the ophthalmic root of the TGG were compared statistically. For the statistical analysis, SPSS ver. 15.0 was used. The mean ± standard deviation of the variables is reported. Since the data showed a normal distribution, intergroup differences were assessed using a one-way ANOVA. The presence of homogeneous variance necessitated the use of the Tukey test for comparisons between two groups. A P<0.05 was accepted as statistically significant.
RESULTS
Two of the animals (n=2) died within the first week and the remaining animals (n=30) were followed for 10 days and then killed. The following clinical findings were frequently observed during the premortem period of the dead animals and the five living animals: signs of meningeal irritation, consciousness, seizures, fever, apnea, cardiac arrhythmia, and breathing disturbances.
TGGs were identified from the trigeminal impressions located on the upper surface of the petrous bones. They were fusiform shaped, and the volumes were estimated to be 2 × 1.5 × 2 mm3 . All trigeminal nerves have three main branches: the ophthalmic, mandibular, and maxillary. The neuron density of only the ophthalmic root division of the TGGs was examined [Figure
Morphological examinations of the brains showed the AChAs at the cisternal segment, extending from its origin to the choroid fissure, and the plexus segment, extending from the choroid fissure to the area where it enters into the choroid plexus of the lateral ventricles. The mean diameter of the AChA examined was 0.35±0.10 mm. The AChA convolutions were more prominent in the animals with severe vasospasm associated with SAH compared to the SHAM, control, and mild vasospasm associated with SAH groups.
To estimate the AChA volume, square-lined glass plates were used and photographs were taken under the microscope during histopathological examinations. The inner elastic membrane (IEM) was less convoluted and the luminal surface area was greater in the SHAM, control [
The mean external diameter/internal diameter of the AChAs using the segmental model was estimated as 115±20 μm/95±20 μm; the mean vasospasm index of the AChAs was 0.46; the mean neuronal density of the ophthalmic root of the TGGs was 8290±1480 neurons/mm3 for all animals (n=32). The mean external diameter/internal diameter of the AChAs was estimated as 140±30 μm/120±30 μm, and the mean vasospasm index of the AChAs was 0.36. The mean neuronal density of the ophthalmic root of the TGGs was 8350±390 neurons/mm 3 in the control group (n=8). The mean external diameter/internal diameter of the AChAs was estimated as 130±20 μm/110±20 μm, and the mean vasospasm index of the AChAs was 0.39. The mean neuronal density of the ophthalmic root of the TGGs was 8550±650 neurons/mm 3 in the SHAM group (n=7). The mean external diameter/internal diameter of the AChAs was estimated as 100±15 μm/80±15 μm, and the mean vasospasm index of the AChAs was 0.56. The mean neuronal density of the ophthalmic root of the TGGs was 8200±600 neurons/mm3 in the SAH group (n=17). Severe vasospasms were observed in seven rabbits with SAH, and mild vasospasms were observed in the remaining 10 rabbits with SAH. The mean external diameter/internal diameter of the AChAs was estimated as 110±20 μm/90±15 μm, and the mean vasospasm index of the AChAs was 0.49. The mean neuronal density of the ophthalmic root of the TGGs was 9800±724 neurons/mm3 for the mild vasospasm with the SAH group (n=10). However, the mean external diameter/internal diameter of the AChAs was estimated as 90±15 μm/60±10 μm, and the mean vasospasm index of the AChAs was 1.25. The mean neuronal density of the ophthalmic root of the TGGs was 6342±557 neurons/mm3 in the severe vasospasm with the SAH group (n=7),
The estimated mean AChAs VSI was significantly higher and the mean neuronal density of the ophthalmic root of the TGG was significantly lower in the severe vasospasm associated with the SAH group when compared to the control, SHAM, and mild vasospasm associated with SAH groups (P< 0.05) [
DISCUSSION
Vasospasm is pathophysiologically characterized by narrowing of the vascular lumen; this may develop following a SAH. Vasospasm after a SAH is associated with significant morbidity and mortality.[
Numerous neuronal, humoral, and chemical factors are involved in cerebrovascular innervations. Parasympathetic fibers have vasodilation effects, and sympathetic fibers have vasospastic effects on cerebral arteries. A variety of autonomic nerve fibers provide neural innervation to cerebral vascular structures. The postganglionic fibers, of the ciliary ganglion, of the third cranial nerve, the sphenopalatine ganglion of the seventh cranial nerve, the otic ganglion of the ninth cranial nerve, and the ganglion of the fifth cranial nerve, are involved in the parasympathetic outflow, which causes vasodilation of the cerebral arteries.[
The extracerebral and intracerebral cranial circulations are innervated by trigeminal nerve fibers. The branches of the ophthalmic division of the trigeminal nerve provide the primary innervations of the large cerebral arteries. The trigeminal nerve fibers release transmitters that cause vasodilation of the cerebral arteries. Direct stimulation of the trigeminal nerve results in ipsilateral cerebral vasodilation after a SAH.[
Vasoactive neurotransmitters, neuromodulators, and neurochemicals are released from trigeminal nerve terminals; these compounds affect large intracranial and extracranial blood vessels.[
The goal of this study was to investigate the potential relationship between the neuron density of the ophthalmic root of the TGG and the severity of AChA vasospasm in rabbits, following a SAH. The two major findings of this investigation were (i) a correlation between the neuron density of the ophthalmic root of the TGG and ACA volume, and (ii) the association of a low neuron density, of the TGG, with the pathogenesis of ACA vasospasm after a SAH. In order to evaluate the severity of AChA vasospasm associated with a SAH, the vasospasm index of the AChA was used as the preferred parameter for assessment rather than the radius of the vessel lumen. As the VSI increased, the degree of arterial vasospasm also increased.
The results of this study showed that the mean AChA VSI was higher in animals with lower neuron density in the trigeminal ganglia. A correlation was found between the neuron density of the ophthalmic division of the trigeminal ganglion and the degree of AChA vasospasm (P<0.05). In particular, the ophthalmic root of the TGG neuron density of the seven rabbits that developed severe vasospasm was significantly lower than in the 10 rabbits that had mild vasospasm. Therefore, a lower neuron density in the TGG might be associated with reduced vasodilation of the AChAs. Compounds associated with vasodilation are synthesized by TGG neurons and secreted from the nerve terminals that end in the cerebral arteries. A reduced number of neurons may result in deficient quantities of vasodilator molecules in the trigeminal ganglia and/or cerebrovascular sensory nerves, which affects the trigemino-cerebrovascular system and the AChAs, and might subsequently increase the severity of vasospasm in the AChAs after a SAH. Therefore, novel approaches to improve cerebral blood flow, under these conditions, are being investigated.
CONCLUSION
The number of neurons in the ophthalmic root of the TGG may play an important role in the regulation of cerebral artery vasospasm (AChAs as well) and, therefore, in the regulation of the cerebral circulation. In rabbits, a significant reduction of the number of the ophthalmic root of the TGG neurons, after a SAH, impaired the sensory innervation of cerebral arteries and the AChAs, and might play an important role in the development of cerebral vasospasm. The results of this study showed that the pathological processes involved in vasospasm, associated with a SAH, significantly affected the choroid arteries. In addition, neurological deficits, resulting in ischemic injury and apoptosis of the choroid plexus, were observed.
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