Vagal Nerve Degeneration and Pulmonary Artery Vasospasm After Subarachnoid Hemorrhage
Mehmet Dumlu AYDIN1, Mehmet Kaan ÜNGÖREN2, Yusuf İZCİ3, Mahmut AÇIKEL4, Cemal GÜNDOĞDU5, Nesrin GÜRSAN5
1Atatürk Üniversitesi Tıp Fakültesi, Nöroşirurji AB, Erzurum, Türkiye
2Lüleburgaz Devlet Hastanesi, Nöroşirurji Servisi, Kırklareli, Türkiye
3Gülhane Askeri Tıp Akademisi, Nöroşirurji AB, Ankara, Türkiye
4Atatürk Üniversitesi Tıp Fakültesi, Kardiyoloji AB, Erzurum, Türkiye
5Atatürk Üniversitesi Tıp Fakültesi, Patoloji AB, Erzurum, Türkiye
Summary
Objectives: The goal of this study is to investigate and demonstrate whether there is a relationship between the ischemic vagal nerve degeneration and pulmonary artery vasospasm in subarachnoid hemorrhage (SAH).
Methods: Animals divided into the 2 groups as control and study groups. In the study group, the animals underwent experimental SAH. All of animals sacrificed and their brains and lungs removed. Then, The vagal nerve (VN) and pulmonary artery (PA) were examined histologically. The degenerated and normal VN axon density and PA vasospasm indexes (VSI) were counted. The values were compered statistically.

Results: Mean VSI of pulmonary arteries was 0,777±0,048, normal VN axon density estimated as 29700±8500/mm2 and degenerated axon density estimated as 30-5/mm2 in control group. In study group, the mean VSI of pulmonary arteries was 1148±0,090, normal VN axon density estimated as 24500±6600/mm2, degenereted axon density estimated as 5300-750/mm2 in slight PA vasospasm detected animals. But, the mean VSI of pulmonary arteries was 1500±0,120, normal VN axon density estimated as 17300±5500/mm2, degenereted axon density estimated as 11300-3860/mm2 in severe PA vasospasm detected animals. Differences were considered to be significant at p< 0.005.

Conclusion: The degenerated VN axon density may be an important factor in the development of PA vasospasm in SAH.

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Methods
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Discussion
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Introduction
Respiratory organs are innervated by somatic and autonomic nervous system. The parasympathetic system has a major role on the continuation of spontaneous respiration and lung pressure regulation(2). The vagal nerve carries postganglionic fibers involved in the autonomic regulation of respiratory organs(3). Autonomic innervation sustained by branches from the vagal nerves and the upper four or five thoracic sympathetic ganglia, all of which contribute nerve fibers to the anterior and posterior pulmonary plexuses at the hilum of each lung(2,9). All afferent nerve fibers to the central nervous system from receptors in the lungs and airways travel in the vagal nevre(11).
Pulmonary hypertension is the most trouble complication of subarachnoid hemorrhages(7,10,12). The lungs are innervated mainly by sympathetic and parasympathetic nerves. Parasympathetic innervation maintained by vasodilatatory fibers of the vagal nerves and vasoconstrictory sympathetic innervation maintained by superior cervical ganglion. Although, subarachnoid hemorrhage causes severely respiratory disturbances and pulmonary hypertension, but has not been investigated the effect of vagal nerve degeneration on pulmonary disorders. Hypertensive lung disease may be possible due to pulmonary artery vasospasm induced by ischemic vagal degeneration in subarachnoid hemorrhage.

We examined whether there is a relationship between the ischemic vagal nerve degeneration and pulmonary artery vasospasm in subarachnoid hemorrhage. The goal of this study is to investigate effects of vagal nerve degeneration on pulmonary disturbances which are cause mortality in SAH. Axonal degeneration of vagal nerve and luminal surface areas of pulmonary arteries were examined histopathologically and between the main surface area values of the pulmonary arteries and the number of degenerated vagal nerve axons were counted and compared statistically.

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Methods
This study was conducted on 19 rabbits. The animal protocols were approved by the Ethics Committee of Atatürk University, Medical Faculty. The care of the animals and the experiments themselves were conducted according to the guidelines of the same committee.
A balanced, injectable anesthetics were used in order reduce pain and mortality. After anesthesia was induced with isoflurane given by a face mask, 0.2 mL/kg of the anesthetic combination (Ketamine HCL, 150 mg/1.5 mL; Xylazine HCL, 30 mg/1.5 mL; and distilled water, 1 mL) was subcutaneously injected before surgery. During the procedure, a dose of 0.1 mL/kg of the anesthetic combination was used when required. Autologous blood (1 mL) was taken from the auricular vein and injected using a 22-Gauge needle into the cisterna magna of animals in the study group over the course of 1 minute. The animals in the control group were not subjected to this procedure. In the control group, 1 mL of serum physiologic was injected into the cisterna magna. The animals were followed for 20 days without any medical treatment and then sacrificed. Whole bodies of all animals were kept in %10 formalin solution after required cleaning procedures for histologic examination.

Five of the rabbits had been selected from baseline control group (n=5). Fourteen of the rabbits were selected from which applied subarachnoid hemorrhage by injecting autologous blood into their cisterna magna, and had been removed their brains and lungs (Figure 1A,B) after twenty days follow up (n=14). The six of fourteen animals selected from have the less vasospasm and have less degenerated vagal axon density included animals (n=6); and eight of them selected from have severe vasospasm and the more degenerated neuron density included animals (n=8).


Click Here to Zoom Figure 1A,B: Macroscopic appearance of brain with SAH (A) and (B) lung in study group.(C2: Arcus of axis, PA: Pulmonary artery, PV: Pulmonary vein)
In order to estimate the axon density of the vagal nerves, all of the vagal nerves together with their ganglions were extracted bilaterally at the levels of under the jugular foramens. Then they were longitudinally embedded in paraffin blocks in order to observe all the roots during the histopathological examination. They were stained with H&E dyes. The Cavalieri method was used to evaluate the density of axons in the vagal nerve sections.(Figure 2A,B) The advantages of this method were that it easily estimated the particle number, could be readily performed, was intuitively simple, was free from assumptions about particle shape, size and orientation and unaffected overprotection and truncation.


Click Here to Zoom Figure 2A,B: Histological examination of the longitudinal vagal nerve sections in control (A) and (B) study group. The axonal loss and expanded interaxonal space are prominent in study group. (NA: Normal axon, DA: Degenerated axon: ScN: Nucleus of Schwann cell) (LM, H&E,x40).
Pulmonary arteries (PA) were obtained from the longitudinal lungs sections at the levels of the 5mm distances from the hilus. They were also stained with H&E dyes. For the calculation of vasospasm index, all PA samples were accepted as a cylinder, in view of their morphological characteristics, and simple geometric formulas were used to estimate their surface areas. As a measure of the degree of vasospasm, the use of PA vasospasm index (VSI) was preferred over the only measurement of lumen radius and volume values because the volume estimation method can be readily performed, is intuitively simple, more reliable, free from assumptions about vessel diameter of various segments and is unaffected by overestimation error of radius values of the PA. PA of all animals were cut 20 segments away from the arising point of the main pulmonary arteries to the entering points of lung tissue. Then, 20 histopathologic sections, 5 µm apart, were obtained by microtome for each designation and are represented by the lines 1,2,3,… and 20. The mean external diameter and internal (luminal) diameters of each section was measured, and external radius was represented as R1 and internal radius was represented as r1. The mean radius value of PA were calculated as R1 = R1 + R2 + R3 +….R20/20; and, lumen radius were calculated as r1 = r1 + r2 + r3 +….r20/20 (Figure 3). The wall ring surface values were calculated as the following formula: S1 = πR12 – πr12. The lumen surface area was calculated as the same method. So, lumen surface value (S2) = πr12. The vasospasm index was calculated as the proportion of S1/S2. Vasospasm Index (VSI) =S1/S2 = πR12 – πr12/ πr12 = π(R12 – r12)/ πr12 = R12 – r12/ r12

In summary, VSI= (R2 – r2)/ r2

The differences between the pulmonary arteries of VSI and axon densities in the vagal nerves were compared statistically. The data were analyzed using a commercially available statistics software package (SPSS® for Windows v. 12.0, Chicago, USA) by author YT.

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Results
Clinically, meningeal irritation signs, consciousness, convulsive attacks, fever, apnea, cardiac arryhytmia and breath disturbances were observed frequently in study group. In normal animals in control group, cardiac pulse was 250±30/min; breath pulse was 30±7 and blood oxygen concentration was %95±5. In the beginning of SAH, the heart rhythm decreased to 140±40/min; breath pulse was 15±5 and blood oxygen concentration was %70±10. A considerable electrocardiographic changes were observed as ST depression, ventricular extrasystols, bigeminal pulses, QRS separation and fibrillations. But in study group, heart pulse was increase in 330±30/min. At the beginning the respiratio rates was 20±4 bpm, about ten hours later it increased to 40±9/min with severe tachypneic&apneic variabilities. In the analyses of respiration parameters, decreased in respiration frequency (Bradypnea) (15±5) and increased respiration amplitude (%30) were observed at the first hours of SAH. Lately, increased respiration frequency (tachypnea) and decreased respiration amplitude (%30±8), shortening inspiration & longing expiration time, apnea-tachypnea attack, diaphragmatic breath and respiration arrest. Macroscopic appearance of a brain is illustrated in Figure 1A; and a normal vagal nerve section is illustrated in Figure 2A. In histopathological examination of vagal nerve, axonal and periaxonal thinning, axonal loss and expanded interaxonal space were accepted as axonal degeneration criterions (Figure 2B). These changes of vagal nerves are less observed in control group in figure 2A and more observed in study group in Figure 2B.
In Stereological examinations, to estimate pulmonary arteries of VSI, squared-lined glass plates were used while photographs were taken under microscope during the histopathological examinations of the PA. The inner elastic membrane (IEM) was less convoluted and the luminal surface area greater in the control and slight vasospastic SAH groups (Figure 3A). The following features were observed in the SAH group: PA narrowing, IEM convolutions, intimal edema formations and endothelial cell shrinkage, desquamation and endothelial cells loss.


Click Here to Zoom Figure 3A,B: Histological examination of the pulmonary arteries in study group. The slight (A) and severe (B) luminal narrowing from pulmonary arteries are prominent in study group.(H: Hemorrhage, Br: Bronchiole). (LM, H&E, x40).
Mean VSI of pulmonary arteries was 0,777±0,048 and normal vagal nerve axon density estimated as 29700±8500/mm3 in control group. The mean VSI of pulmonary arteries was 1,148±0,090 and normal vagal nerve axon density estimated as 24500±6600/mm3 in slight pulmonary artery vasospasm detected animals in study group. But, the mean VSI of pulmonary arteries was 1,500±0,120 and normal vagal nerve axon density estimated as 17300±5500/mm3 in severe pulmonary artery vasospasm detected animals in study group (Figure 3B). The relationship between the degenerated vagal axon density and the severity of pulmonary artery vasospasm was found important by statistically. In comparison of the respiration parameters and histopathological changes of the vagal nerves, the following results were drawn: Degenerated axons numbers of vagal nerves were more prominent in the severe PA spasm developed animals (Table 1). The data analysis consisted of the Kruskal-Wallis and Mann-Whitney U test. Differences were considered to be significant at p< 0.05.


Click Here to Zoom Table 1: Degenerated and normal axons numbers of vagal nerves and VSI values of pulmonary artery
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Discussion
Respiration can be defined as a process of gas exchange in the lungs. Respiratory organs are innervated by vagal nerves, sympathetic nerves and the upper four or five thoracic somatic nerves(5,13). All nerves forms pulmonary ganglia composed of excitatory cholinergic neurons and inhibitory noradrenergic neurons and somatosensitive neurons. All autonomic and somatic nerves control the calibers of the conducting airways and pulmonary blood vessels, the volume of respiratory units, the activity of bronchial glands and respiration reflexes(1,4). The origins of sensory innervation of the lower respiratory tract are thought to be principally the nodose and jugular ganglia of the vagus nerve, spinal ganglia at levels C2-C6 and T1-T6 ganglia. Impulses of chemoreceptors are conveyed by glossopharyngeal and vagal nerve fibers to the respiration centers. The continuation of pulmonary based reflexes are maintained prominently by vagal nerves(2,11). Vagal pulmonary myelinated afferents are innervate lung tissue and pulmonary neurones of vagal nerves are localised in nodose ganglia(14). Vagal nerve is the major principle neural pathway which interconnects the brainstem and the lungs and involved in both the central respiration rhythm, regulation of pulmonary vasculature, airways and pulmonary secretion(8,15). It is well known that the vagal nerves play many important roles on the maintenance of respiration such as regulation of airways and pulmonary vessels resistance, pulmonary pressure, Hering-Breuer reflex, blood pH, respiration and heart rhythm coordination, lung metabolism and immunity.
Pulmonary hypertension is the most trouble complication of subarachnoid hemorrhages and it is characterized by constructive and complex arterial lesions affecting the pulmonary arteries. However, not only vagal nerves but also glossopharyngeal and other lower cranial nerves and upper cervical spinal nerves are also injured due to subarachnoid hemorrhage extending into cervical spinal canal and aggravates the mortal effects of SAH. Ischemic vagal nerves induced by SAH generate uncontrollable parasympathetic discharges an rely on lung edema via dilated PA. Also, overdyscharges of vagal nerves may be responsible for increased cholinergic activity on the heart and disordered heart functions at the beginning of SAH. On the contrary, it was shown that the undyscharged ischemic vagal nerves could rely on heart rhythm variability and cardiac arrest in the late phase of subarachnoid hemorrhage(6). By the similar mechanisms, ischemic vagal nerve roots can not drive the parasympathetic stimulation the PA and heart; and, both untreatable PA vasospasm and uncontrollable heart rhythm variabilities can be inevitable in serious SAH. Parasympathetic vasodilatatory impulses of vagal nerves have major roles on the continuation of PA circulation in the normal limits. Ischemic injury of vagal nerve complexes induced by SAH can block the parasympathetic controls on the lungs and heart. This pathophysiologic processes invite indirectly increased sympathetic hyperactivity. Decreased parasympathetic and increased sympathetic impulses triggers the development of both massive lung edema, PA vasospasm and depleted the heart reserves. We proposed that the subcutaneous vagal nerve blockages may be useful at the beginning of SAH; vagal nerve stimulation or sympathetic blockages may be useful at the late phases of SAH. We suggested that SAH resulted in vagal nerve ischemia at the brainstem due to both afferent and efferent vagal nerve root supplying arteries vasospasm, and degenerated vagal ganglions neuron density may play major roles on the development of pulmonary arteries vasospasm. The degenerated axon density of vagal nerves were found in the less levels in the slight PA vasospasm developed animals. But, the degenerated axon density of vagal nerves were found in the high levels in the severe PA vasospasm developed animals.

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Conclusion
Vagal nerves regulate the respiration cycle and pulmonary circulation pressure. When the vagal nerves are injured, pulmonary artery vasospasm may occur secondary to pulmonary hypertension. Consequently respiration disturbances may be developed by vagal nerve injury at the brainstem level after progressive and fatal subarachnoid hemorrhage.
Received by: 01 May 2012
Revised by: 04 February 2013
Accepted: 11 April 2013

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References
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