The Role of Pterygopalatine Ganglion Neuron Density in the Severity of APA Vasospasm Following Subarachnoid Hemorrhage: A Preliminary Experimental Study

The Role of Pterygopalatine Ganglion Neuron Density in the Severity of APA Vasospasm Following Subarachnoid Hemorrhage: A Preliminary Experimental Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Role of Pterygopalatine Ganglion Neuron Density in the Severity of APA Vasospasm Following Subarachnoid Hemorrhage: A Preliminary Experimental Study Caner Fahrettin Kara, Muhammet Elveren, Yakup Cağlaroğlu, Ufuk Temtek, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6253817/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Anterior perforating arteries (APA) play a critical role in cerebral circulation. Whether there is a correlation between APA vasospasm and the neuronal density of the pterygopalatine ganglion during subarachnoid hemorrhage (SAH) has not yet been elucidated. Methods: This study included 21 rabbits, divided into three groups: control (n=5), sham (n=5), and SAH (n=11). SAH was induced by injecting autologous blood into the cisterna magna. Seven days post-induction, the animals were decapitated, and both the APA and pterygopalatine ganglia were stereologically analyzed. Vasospasm severity was quantified using the vasospasm index (VSI). VSI values and neuronal densities were statistically evaluated using the Kruskal-Wallis and Mann-Whitney U tests, with significance set at p < 0.005. Results: Significant differences were found among the groups in both VSI values and neuronal densities. The control group exhibited a VSI of 0.234 ± 0.031, the sham group 0.995 ± 0.121, and the SAH group showed 1.127 ± 0.654 in cases of mild vasospasm and 2.126 ± 0.986 in cases of severe vasospasm. Neuronal density was also significantly reduced in the SAH group compared to the control group. Conclusion: The neuronal density of the pterygopalatine ganglion may significantly influence the severity of APA vasospasm following subarachnoid hemorrhage. Pterygopalatine ganglion anterior perforating arteries subarachnoid hemorrhage vasospasm Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cerebral vasospasm remains one of the most challenging complications following subarachnoid hemorrhage (SAH) [ 1 ]. Within this complex clinical scenario, a key structure is the anterior perforating artery (APA), a critical branch of the supraclinoid segment of the internal carotid artery (ICA) [ 2 ]. The innervation of the APA reflects a delicate balance between parasympathetic fibers, which mediate vasodilation, and sympathetic fibers, which promote vasospasm. Parasympathetic outflow is primarily mediated by postganglionic fibers arising from the sphenopalatine ganglion (cranial nerve VII) and the otic ganglion, which receives preganglionic input from the glossopharyngeal nerve (cranial nerve IX) [ 3 ]. In contrast, cerebral vessels also receive vasoconstrictive sympathetic postganglionic fibers originating from the superior cervical ganglion [ 4 ]. The central sympathetic system plays a pivotal role in the pathogenesis of vasospasm after SAH [ 5 ]. Neuronal density within peripheral nerve ganglia significantly influences these vasomotor functions [ 6 ]. Notably, a marked reduction in parasympathetic nerve density has been observed in the arteries of the anterior circulation in patients with Alzheimer’s disease [ 7 ]. Experimental evidence has shown that electrical stimulation of the pterygopalatine ganglion (PPG) induces vasodilation in ipsilateral cerebral arteries, while its ablation via cauterization results in vasoconstriction [ 8 ]. The vasospasm index (VSI) of the APA is defined as the ratio between the vessel wall area and the luminal area, highlighting the relationship between ganglionic neuronal density and vasospasm. A lower neuronal density in the PPG is associated with increased APA vasospasm, underscoring the critical role of neuronal control in cerebrovascular regulation. Materials and Methods Animal Selection and Experimental Groups: This study included 21 New Zealand rabbits aged 2 ± 0.1 years and weighing 4 ± 0.5 kg. All animals were obtained from the Atatürk University Medical Experimental Application and Research Center (Erzurum, Turkey), a licensed and accredited animal research facility operating under the regulations of the Turkish Ministry of Agriculture and Forestry. No animals were sourced from private individuals or institutions. Ethical approval was obtained from the Animal Experiments Ethics Committee of the Faculty of Medicine, Atatürk University. The animals were randomly divided into three groups: control (n = 5), sham (n = 5), and experimental (n = 11). Both the sham and experimental groups initially received subcutaneous anesthesia with 0.2 mL/kg isoflurane and 50 mg/1.5 mL ketamine HCl. General anesthesia was maintained with 30 mg/1.5 mL xylazine HCl and 1 mL of distilled water. After preparing the occipito-cervical region under sterile conditions, the animals’ heads were hyperflexed, and the cisterna magna was accessed through the posterior notch of the foramen magnum. After aspirating 1 cc of cerebrospinal fluid to confirm the absence of bleeding, 0.5 cc of isotonic saline was injected into the cisterna magna in the sham group, while 0.5 cc of autologous blood obtained from the auricular artery was injected in the experimental group. Vital signs were monitored twice daily throughout the experiment. Three weeks later, following intracardiac injection of 2 cc of formalin, the animals were decapitated at the level of the fifth cervical vertebra and incubated in formalin solution for one week. Subsequently, the brains, along with the facial and glossopharyngeal nerves and the pterygopalatine ganglia, were carefully dissected under an operating microscope and fixed in 10% formalin for five days. Histopathological Procedures: The brains were sectioned to include the anterior perforated area and embedded horizontally in paraffin blocks. Twenty sections were obtained at 5 µm intervals along the trajectory of the APA, preserving microanatomical and histological structures. Tissues containing branches of the facial and glossopharyngeal nerves and the pterygopalatine ganglia were also embedded in paraffin. Five-micron sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope. Images were captured at 20× and 40× magnifications. The Cavalieri method was used to evaluate the neuronal density of the pterygopalatine ganglion [ 9 ]. Stereological Analysis: Stereological methods provide reliable estimates of the number and volume of cells and particles. This study utilized stereological techniques from prior research to estimate PPG neuronal density [ 6 ]. The physical dissector method was employed to quantify the number of normal and degenerative neurons in the PPG, allowing for the accurate estimation of neuronal density. Differences in normal and degenerative neuronal densities were statistically compared. Criteria for neuronal degeneration included angular cell body deformation, cytoplasmic condensation, nuclear shrinkage, and apoptotic changes. The severity of vasospasm in the APA was estimated using the vasospasm index (VSI), calculated as the ratio of vessel wall surface area to lumen area using the formula: (R² − r²)/r². Statistical Analysis: All data were analyzed using SPSS® for Windows, version 12.0 (Chicago, USA). The Kruskal–Wallis and Mann–Whitney U tests were applied, and statistical significance was set at p < 0.05. Results Clinical Findings: During the first week, three animals from the experimental group (n = 2) and one from the sham group (n = 1) died. Observed symptoms in the experimental group included neck stiffness, loss of consciousness, seizures, fever, apnea, cardiac arrhythmias, and respiratory dysfunction. Electrocardiographic (ECG) recordings revealed ventricular extrasystoles, ST segment depression, QRS dissociation, bigeminal or trigeminal extrasystoles, and atrioventricular fibrillation. Gross Pathological Findings: Postmortem dissection revealed thickening and adhesions on the brain surface, hyperemia, edematous appearance, and clot formation. The arachnoid and pia mater membranes covering the basal cisterns showed obliteration of the sulci. Histopathological Findings: Quantitative and Statistical Analysis Numerical Results: This study analyzed the neuronal density of the pterygopalatine ganglion and the vasospasm index (VSI) across different groups. The mean values of normal and degenerative neuron densities, along with VSI measurements, were calculated as follows. Table 1 Mean normal and degenerative neuron densities, and vasospasm index (VSI) values for each group. Group Mean Normal Neuron Density (n/mm³) Mean Degenerative Neuron Density (n/mm³) Vasospasm Index (VSI) Control Group (n = 5) 12,310 ± 1,590 12 ± 3 0.234 ± 0.031 SHAM Group (n = 5) 12,165 ± 1,370 56 ± 8 0.995 ± 0.121 Experimental Group A (n = 6) 10,110 ± 1,170 782 ± 112 1.127 ± 0.654 Experimental Group B (n = 5) 8,960 ± 990 1,954 ± 189 2.126 ± 0.986 Statistical Results: Statistical analysis revealed significant differences among the groups in both neuronal density and vasospasm index (VSI) values. Table 2 :Statistical comparisons of mean neuronal and degenerative neuron densities, and VSI values between groups.. Comparison p value Statistical significance Control vs. SHAM p < 0.005 Statistically significant difference SHAM vs. Experimental Group p < 0.0005 Highly significant difference Control vs. Experimental Group p < 0.000001 Extremely significant difference These results demonstrate a stepwise increase in neuronal degeneration and VSI values from the control group through the sham group to the experimental groups. Discussion The results of this study clearly demonstrate the relationship between anterior perforating artery (APA) vasospasm and neuronal density in the pterygopalatine ganglion (PPG). Cerebral vasospasm following subarachnoid hemorrhage (SAH) is a major source of morbidity and mortality. Using an experimental SAH model, this study examined the influence of neuronal density in the PPG on APA vasospasm severity. Our findings are consistent with existing literature that highlights the roles of the sympathetic and parasympathetic systems in the pathophysiology of cerebral vasospasm. The PPG receives preganglionic parasympathetic fibers from the superior salivatory nucleus via the facial nerve, the greater superficial petrosal nerve, and the Vidian nerve through the pterygopalatine canal nerve [ 10 , 11 ]. Postganglionic fibers from the ganglion innervate intracranial extracerebral blood vessels, the lacrimal gland, the forehead, and the nasal mucosa. These fibers release vasoactive substances such as nitric oxide, vasoactive intestinal peptide (VIP), and acetylcholine, thereby inducing vasodilation [ 12 , 13 ]. Stimulation of the sphenopalatine ganglion (SPG) has been shown to increase ipsilateral cerebral blood flow in cats and rats, with a 15–20% enlargement in the diameter of major arteries in the circle of Willis following stimulation [ 11 , 14 ]. Cerebral vasospasm is a critical cause of morbidity after SAH, primarily affecting large-capacity cerebral arteries [ 1 ]. The morphology of the PPG plays a significant role in these vascular changes and exerts its influence through peripheral pathways [ 6 ]. A reduction in parasympathetic input from the PPG can exacerbate vasospasm [ 15 ]. SPG stimulation promotes vasodilation in the arteries of the circle of Willis and increases cerebrospinal fluid (CSF) production, thus reducing vasospasm and enhancing blood–brain barrier permeability [ 16 ]. In parallel with our findings, experimental groups showed evident APA vasospasm and marked neuronal degeneration in the PPG. In particular, Group B (severe vasospasm) exhibited a significant reduction in normal neuronal density and a prominent increase in degenerative neurons. Compared to the control group, even the sham group demonstrated decreased neuronal density and increased VSI. These findings support the critical role of PPG neuronal density in regulating cerebral vascular tone. Modulation of the PPG has also been employed in the treatment of conditions such as migraine, cluster headache, and trigeminal neuralgia [ 17 ]. Electrical stimulation of the SPG has been found to alleviate vasospasm in experimental models of SAH [ 16 ]. This study provides valuable insights into potential therapeutic strategies for managing cerebral vasospasm following SAH. In particular, modulation of the PPG or its downstream neural pathways may represent a promising target for both prevention and treatment of cerebral vasospasm. The reduced parasympathetic nerve density observed in patients with Alzheimer’s disease may also contribute to the development of cerebrovascular disorders [ 7 ]. SPG stimulation has been shown to reduce vasospasm by increasing the diameter of arteries within the circle of Willis [ 11 , 14 ]. Additional experimental studies suggest that SPG stimulation may have further beneficial effects following SAH. Yarnitsky et al. reported that SPG stimulation increased cerebral blood flow (CBF), reduced infarct size, and improved neurological function following permanent middle cerebral artery (MCA) occlusion. Henninger and Fisher demonstrated a reduction in infarct volume associated with improved perfusion in penumbral regions. Other neuromodulatory approaches, such as stimulation of the fastigial nucleus, have been shown to reduce neurogenic inflammation, spreading depression, and neuronal apoptosis, offering protection following cerebral ischemia [ 14 , 18 , 19 ]. Limitations and Future Directions This study has several limitations. The experimental model may not fully replicate human physiology, and the sample size was relatively small. Future studies should aim to validate these findings using larger sample sizes and various animal models. In addition, advanced translational research is needed to assess the clinical efficacy of PPG modulation. Conclusion In conclusion, this study elucidates the complex relationship between APA vasospasm and PPG neuronal density, contributing to the development of novel approaches for the prevention and treatment of cerebral vasospasm. Declarations Ethical Approval This study was approved by the Clinical Research Ethics Committee of Atatürk University (Approval No: 2200369130, Date: 09.11.2022). All animal care and experimental procedures were conducted in accordance with institutional guidelines approved by the same committee. Compliance with guidelines: All procedures performed in this study involving animals complied with national and international guidelines, including the Basel Declaration and the principles outlined by Animal Research Tomorrow ( https://animalresearchtomorrow.org/en ). Author Contribution M.E. Y.C. U.T and C.K. wrote the main text of the manuscript, contributed to the experimental part of the study, and performed the statistical calculations. A.T and E.D. prepared the pathological samples and created figures 1-8. All authors contributed to the study design, data analysis, and manuscript revision, and reviewed and approved the final manuscript. Acknowledgement The authors would like to express their sincere gratitude to Prof. Dr. Dumlu Aydın from the Department of Neurosurgery, Atatürk University Faculty of Medicine, for his valuable guidance in obtaining ethical approval and for his insightful contributions to the interpretation and evaluation of the study findings. Data Availability The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. 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Aydin, M.D., et al., The role of neuron numbers of the petrosal ganglion in the determination of blood pressure: an experimental study . Minim Invasive Neurosurg, 2006. 49(6): p. 359–61. Bleys, R.L., et al., Perivascular nerves of the human basal cerebral arteries: II. Changes in aging and Alzheimer's disease . J Cereb Blood Flow Metab, 1996. 16(5): p. 1048–57. Toda, N., et al., Preganglionic and postganglionic neurons responsible for cerebral vasodilation mediated by nitric oxide in anesthetized dogs . J Cereb Blood Flow Metab, 2000. 20(4): p. 700–8. Gundersen, H.J., et al., Some new, simple and efficient stereological methods and their use in pathological research and diagnosis . Apmis, 1988. 96(5): p. 379–94. Hara, H., et al., Acetylcholine and vasoactive intestinal peptide in cerebral blood vessels: effect of extirpation of the sphenopalatine ganglion . J Cereb Blood Flow Metab, 1989. 9(2): p. 204–11. Toda, N., et al., Cerebral vasodilatation induced by stimulation of the pterygopalatine ganglion and greater petrosal nerve in anesthetized monkeys . Neuroscience, 2000. 96(2): p. 393–8. Edvinsson, L., et al., VIP (vasoactive intestinal polypeptide)-containing nerves of intracranial arteries in mammals . Cell Tissue Res, 1980. 208(1): p. 135–42. Edvinsson, L. and D.N. Krause, Cerebral blood flow and metabolism . 2002: Lippincott Williams & Wilkins. Yarnitsky, D., et al., Reversal of cerebral vasospasm by sphenopalatine ganglion stimulation in a dog model of subarachnoid hemorrhage . Surg Neurol, 2005. 64(1): p. 5–11; discussion 11. Boysen, N.C., D.N. Dragon, and W.T. Talman, Parasympathetic tonic dilatory influences on cerebral vessels . Auton Neurosci, 2009. 147(1–2): p. 101–4. Takahashi, M., Z.D. Zhang, and R.L. Macdonald, Sphenopalatine ganglion stimulation for vasospasm after experimental subarachnoid hemorrhage . J Neurosurg, 2011. 114(4): p. 1104–9. Ho, K.W.D., R. Przkora, and S. Kumar, Sphenopalatine ganglion: block, radiofrequency ablation and neurostimulation - a systematic review . J Headache Pain, 2017. 18(1): p. 118. Galea, E., et al., Stimulation of cerebellar fastigial nucleus inhibits interleukin-1beta-induced cerebrovascular inflammation . Am J Physiol, 1998. 275(6): p. H2053-63. Reis, D.J., et al., Brief electrical stimulation of cerebellar fastigial nucleus conditions long-lasting salvage from focal cerebral ischemia: conditioned central neurogenic neuroprotection . Brain Res, 1998. 780(1): p. 161–5. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6253817","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472436620,"identity":"15e424e8-57ef-4713-ae29-c934297c1927","order_by":0,"name":"Caner Fahrettin Kara","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIie3PMUvDQBTA8QuBToHb5IKQfIV3BFqkol8lR4Yuqa4FBZ9LOna1+D2C44WDupxkPXCSQqcM6SJ2ERvrIiSl3TrcfzhueD8ejxCb7WSTzeNKEhO3+aA8kPTiP+IcTDxoNvySvcN0+lYsJ/rqlk71J/t4GQZQFo/Km5CQnrUvY/om4dokF096nDOhRxEYgcrThM+f41YCJO37WLtA5Dg3IlMiNw4W84zE8N5BaDXYYP0AYVmtdqQstuR7D2Fp30GjAEza2xEpUK6xmzBTRT7qV+BmNfgS2Si63t4i6wXrvIXOUr7GxR0EZbLkm2wY+DOl6vj+MqTn7aQzdty4zWaz2f71A1KQa85fwUsnAAAAAElFTkSuQmCC","orcid":"","institution":"giresun","correspondingAuthor":true,"prefix":"","firstName":"Caner","middleName":"Fahrettin","lastName":"Kara","suffix":""},{"id":472436621,"identity":"569a9eec-a06f-4160-b921-55b24777a1f2","order_by":1,"name":"Muhammet Elveren","email":"","orcid":"","institution":"Erzurum Regional Training and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Muhammet","middleName":"","lastName":"Elveren","suffix":""},{"id":472436622,"identity":"e92f2192-cb5c-4d6a-b439-177aeba08735","order_by":2,"name":"Yakup Cağlaroğlu","email":"","orcid":"","institution":"Erzurum Regional Training and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yakup","middleName":"","lastName":"Cağlaroğlu","suffix":""},{"id":472436623,"identity":"794c7a4b-f58c-4266-8d9b-4663d374530c","order_by":3,"name":"Ufuk Temtek","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Ufuk","middleName":"","lastName":"Temtek","suffix":""},{"id":472436624,"identity":"8a51ab5f-17dd-4714-9e12-0f8503548480","order_by":4,"name":"Aysenur Temtek","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Aysenur","middleName":"","lastName":"Temtek","suffix":""},{"id":472436625,"identity":"543709fa-c66b-4f42-86d8-f295166ec5f2","order_by":5,"name":"Elif Demirci","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Elif","middleName":"","lastName":"Demirci","suffix":""}],"badges":[],"createdAt":"2025-03-18 13:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6253817/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6253817/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85177327,"identity":"95f38432-984c-4ac4-943c-e5573c702118","added_by":"auto","created_at":"2025-06-23 06:43:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149809,"visible":true,"origin":"","legend":"\u003cp\u003eNormal APA in a rabbit (LM, H\u0026amp;E, ×4/A; ×10/B).\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6253817/v1/4d7ec3431ed7081c0610e4aa.jpeg"},{"id":85176167,"identity":"76d8264c-ec5c-44e8-81ae-f33208044b25","added_by":"auto","created_at":"2025-06-23 06:35:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101598,"visible":true,"origin":"","legend":"\u003cp\u003eMinimally narrowed APA in a sham rabbit (LM, H\u0026amp;E, ×4/A; ×10/B).\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6253817/v1/4d88ab7059c0576a70929462.jpg"},{"id":85176170,"identity":"a349faaf-2725-4497-b0bb-6f7c9f408d2f","added_by":"auto","created_at":"2025-06-23 06:35:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180067,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental subject showing numerous degenerative neurons in the PPG, with prominent cellular changes and inflammation (LM, H\u0026amp;E, ×4/A; ×20/B).\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6253817/v1/a0a8fa59c3404e3aa2f51637.jpeg"},{"id":85177783,"identity":"c507956d-e1f4-4d1d-b41d-eb54eabd0d66","added_by":"auto","created_at":"2025-06-23 06:51:14","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":134296,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental subject with moderate neuronal degeneration in the PPG (LM, H\u0026amp;E, ×4/A; ×20/B).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6253817/v1/57500a84a5dece8177f9a807.jpeg"},{"id":85176175,"identity":"61b4dd67-6203-4ed6-a0ea-6baaaba47935","added_by":"auto","created_at":"2025-06-23 06:35:14","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":193977,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental subject with minimal neuronal degeneration in the PPG (LM, H\u0026amp;E, ×4/A; ×20/B)\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6253817/v1/121c790e77fc7bb8f2d6e640.jpeg"},{"id":85177334,"identity":"9e85f419-b815-40ef-87e0-4863b61c2e93","added_by":"auto","created_at":"2025-06-23 06:43:14","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":137768,"visible":true,"origin":"","legend":"\u003cp\u003eSeverely degenerative PPG neurons (LM, Aldehyde fuchsin, ×50/A) and associated vascular changes (LM, H\u0026amp;E, ×4/A; ×20/B).\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6253817/v1/af7a837e75ef4ae49db90fc3.jpeg"},{"id":85176182,"identity":"6069cea4-ff03-4bf1-a081-371097d5511c","added_by":"auto","created_at":"2025-06-23 06:35:15","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":119963,"visible":true,"origin":"","legend":"\u003cp\u003eMethod used for calculating the vasospasm index (VSI) (LM, H\u0026amp;E, ×40).\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6253817/v1/2ecd6062cc3fec356ea82e7b.jpeg"},{"id":85176177,"identity":"818a93eb-ca5c-4ed0-8fd4-e9507da79cee","added_by":"auto","created_at":"2025-06-23 06:35:14","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":36014,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical correlation between degenerative neuron density (n/mm³) and VSI values.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6253817/v1/05a766241041ba9c677f6be1.jpeg"},{"id":91115091,"identity":"d7a5df94-470a-44a9-a5cf-9e7361bf1854","added_by":"auto","created_at":"2025-09-11 17:16:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1694068,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6253817/v1/d15cbca9-1806-4f67-8f43-30224ed6fbb1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Role of Pterygopalatine Ganglion Neuron Density in the Severity of APA Vasospasm Following Subarachnoid Hemorrhage: A Preliminary Experimental Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCerebral vasospasm remains one of the most challenging complications following subarachnoid hemorrhage (SAH) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Within this complex clinical scenario, a key structure is the anterior perforating artery (APA), a critical branch of the supraclinoid segment of the internal carotid artery (ICA) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The innervation of the APA reflects a delicate balance between parasympathetic fibers, which mediate vasodilation, and sympathetic fibers, which promote vasospasm. Parasympathetic outflow is primarily mediated by postganglionic fibers arising from the sphenopalatine ganglion (cranial nerve VII) and the otic ganglion, which receives preganglionic input from the glossopharyngeal nerve (cranial nerve IX) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In contrast, cerebral vessels also receive vasoconstrictive sympathetic postganglionic fibers originating from the superior cervical ganglion [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe central sympathetic system plays a pivotal role in the pathogenesis of vasospasm after SAH [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Neuronal density within peripheral nerve ganglia significantly influences these vasomotor functions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Notably, a marked reduction in parasympathetic nerve density has been observed in the arteries of the anterior circulation in patients with Alzheimer\u0026rsquo;s disease [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Experimental evidence has shown that electrical stimulation of the pterygopalatine ganglion (PPG) induces vasodilation in ipsilateral cerebral arteries, while its ablation via cauterization results in vasoconstriction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe vasospasm index (VSI) of the APA is defined as the ratio between the vessel wall area and the luminal area, highlighting the relationship between ganglionic neuronal density and vasospasm. A lower neuronal density in the PPG is associated with increased APA vasospasm, underscoring the critical role of neuronal control in cerebrovascular regulation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Selection and Experimental Groups:\u003c/h2\u003e \u003cp\u003eThis study included 21 New Zealand rabbits aged 2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 years and weighing 4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 kg. All animals were obtained from the Atat\u0026uuml;rk University Medical Experimental Application and Research Center (Erzurum, Turkey), a licensed and accredited animal research facility operating under the regulations of the Turkish Ministry of Agriculture and Forestry. No animals were sourced from private individuals or institutions. Ethical approval was obtained from the Animal Experiments Ethics Committee of the Faculty of Medicine, Atat\u0026uuml;rk University. The animals were randomly divided into three groups: control (n\u0026thinsp;=\u0026thinsp;5), sham (n\u0026thinsp;=\u0026thinsp;5), and experimental (n\u0026thinsp;=\u0026thinsp;11). Both the sham and experimental groups initially received subcutaneous anesthesia with 0.2 mL/kg isoflurane and 50 mg/1.5 mL ketamine HCl. General anesthesia was maintained with 30 mg/1.5 mL xylazine HCl and 1 mL of distilled water. After preparing the occipito-cervical region under sterile conditions, the animals\u0026rsquo; heads were hyperflexed, and the cisterna magna was accessed through the posterior notch of the foramen magnum. After aspirating 1 cc of cerebrospinal fluid to confirm the absence of bleeding, 0.5 cc of isotonic saline was injected into the cisterna magna in the sham group, while 0.5 cc of autologous blood obtained from the auricular artery was injected in the experimental group. Vital signs were monitored twice daily throughout the experiment. Three weeks later, following intracardiac injection of 2 cc of formalin, the animals were decapitated at the level of the fifth cervical vertebra and incubated in formalin solution for one week. Subsequently, the brains, along with the facial and glossopharyngeal nerves and the pterygopalatine ganglia, were carefully dissected under an operating microscope and fixed in 10% formalin for five days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathological Procedures:\u003c/h3\u003e\n\u003cp\u003eThe brains were sectioned to include the anterior perforated area and embedded horizontally in paraffin blocks. Twenty sections were obtained at 5 \u0026micro;m intervals along the trajectory of the APA, preserving microanatomical and histological structures. Tissues containing branches of the facial and glossopharyngeal nerves and the pterygopalatine ganglia were also embedded in paraffin. Five-micron sections were stained with hematoxylin and eosin (H\u0026amp;E) and examined under a light microscope. Images were captured at 20\u0026times; and 40\u0026times; magnifications. The Cavalieri method was used to evaluate the neuronal density of the pterygopalatine ganglion [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eStereological Analysis:\u003c/h3\u003e\n\u003cp\u003eStereological methods provide reliable estimates of the number and volume of cells and particles. This study utilized stereological techniques from prior research to estimate PPG neuronal density [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The physical dissector method was employed to quantify the number of normal and degenerative neurons in the PPG, allowing for the accurate estimation of neuronal density. Differences in normal and degenerative neuronal densities were statistically compared. Criteria for neuronal degeneration included angular cell body deformation, cytoplasmic condensation, nuclear shrinkage, and apoptotic changes. The severity of vasospasm in the APA was estimated using the vasospasm index (VSI), calculated as the ratio of vessel wall surface area to lumen area using the formula: (R\u0026sup2; \u0026minus; r\u0026sup2;)/r\u0026sup2;.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eAll data were analyzed using SPSS\u0026reg; for Windows, version 12.0 (Chicago, USA). The Kruskal\u0026ndash;Wallis and Mann\u0026ndash;Whitney U tests were applied, and statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eClinical Findings:\u003c/h2\u003e\n \u003cp\u003eDuring the first week, three animals from the experimental group (n\u0026thinsp;=\u0026thinsp;2) and one from the sham group (n\u0026thinsp;=\u0026thinsp;1) died. Observed symptoms in the experimental group included neck stiffness, loss of consciousness, seizures, fever, apnea, cardiac arrhythmias, and respiratory dysfunction. Electrocardiographic (ECG) recordings revealed ventricular extrasystoles, ST segment depression, QRS dissociation, bigeminal or trigeminal extrasystoles, and atrioventricular fibrillation.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eGross Pathological Findings:\u003c/h3\u003e\n\u003cp\u003ePostmortem dissection revealed thickening and adhesions on the brain surface, hyperemia, edematous appearance, and clot formation. The arachnoid and pia mater membranes covering the basal cisterns showed obliteration of the sulci.\u003c/p\u003e\n\u003ch3\u003eHistopathological Findings:\u0026nbsp;\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eQuantitative and Statistical Analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003eNumerical Results:\u003c/h2\u003e\n \u003cp\u003eThis study analyzed the neuronal density of the pterygopalatine ganglion and the vasospasm index (VSI) across different groups. The mean values of normal and degenerative neuron densities, along with VSI measurements, were calculated as follows.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMean normal and degenerative neuron densities, and vasospasm index (VSI) values for each group.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean Normal Neuron Density (n/mm\u0026sup3;)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean Degenerative Neuron Density (n/mm\u0026sup3;)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVasospasm Index (VSI)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl Group (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12,310\u0026thinsp;\u0026plusmn;\u0026thinsp;1,590\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.234\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSHAM Group (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12,165\u0026thinsp;\u0026plusmn;\u0026thinsp;1,370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.995\u0026thinsp;\u0026plusmn;\u0026thinsp;0.121\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExperimental Group A (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10,110\u0026thinsp;\u0026plusmn;\u0026thinsp;1,170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e782\u0026thinsp;\u0026plusmn;\u0026thinsp;112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.127\u0026thinsp;\u0026plusmn;\u0026thinsp;0.654\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExperimental Group B (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8,960\u0026thinsp;\u0026plusmn;\u0026thinsp;990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1,954\u0026thinsp;\u0026plusmn;\u0026thinsp;189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.126\u0026thinsp;\u0026plusmn;\u0026thinsp;0.986\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Results:\u003c/h2\u003e\n \u003cp\u003eStatistical analysis revealed significant differences among the groups in both neuronal density and vasospasm index (VSI) values.\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e:Statistical comparisons of mean neuronal and degenerative neuron densities, and VSI values between groups..\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eComparison\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStatistical significance\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl vs. SHAM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStatistically significant difference\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSHAM vs. Experimental Group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHighly significant difference\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl vs. Experimental Group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.000001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExtremely significant difference\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThese results demonstrate a stepwise increase in neuronal degeneration and VSI values from the control group through the sham group to the experimental groups.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study clearly demonstrate the relationship between anterior perforating artery (APA) vasospasm and neuronal density in the pterygopalatine ganglion (PPG). Cerebral vasospasm following subarachnoid hemorrhage (SAH) is a major source of morbidity and mortality. Using an experimental SAH model, this study examined the influence of neuronal density in the PPG on APA vasospasm severity.\u003c/p\u003e \u003cp\u003eOur findings are consistent with existing literature that highlights the roles of the sympathetic and parasympathetic systems in the pathophysiology of cerebral vasospasm. The PPG receives preganglionic parasympathetic fibers from the superior salivatory nucleus via the facial nerve, the greater superficial petrosal nerve, and the Vidian nerve through the pterygopalatine canal nerve [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Postganglionic fibers from the ganglion innervate intracranial extracerebral blood vessels, the lacrimal gland, the forehead, and the nasal mucosa. These fibers release vasoactive substances such as nitric oxide, vasoactive intestinal peptide (VIP), and acetylcholine, thereby inducing vasodilation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Stimulation of the sphenopalatine ganglion (SPG) has been shown to increase ipsilateral cerebral blood flow in cats and rats, with a 15\u0026ndash;20% enlargement in the diameter of major arteries in the circle of Willis following stimulation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCerebral vasospasm is a critical cause of morbidity after SAH, primarily affecting large-capacity cerebral arteries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The morphology of the PPG plays a significant role in these vascular changes and exerts its influence through peripheral pathways [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A reduction in parasympathetic input from the PPG can exacerbate vasospasm [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. SPG stimulation promotes vasodilation in the arteries of the circle of Willis and increases cerebrospinal fluid (CSF) production, thus reducing vasospasm and enhancing blood\u0026ndash;brain barrier permeability [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In parallel with our findings, experimental groups showed evident APA vasospasm and marked neuronal degeneration in the PPG. In particular, Group B (severe vasospasm) exhibited a significant reduction in normal neuronal density and a prominent increase in degenerative neurons. Compared to the control group, even the sham group demonstrated decreased neuronal density and increased VSI.\u003c/p\u003e \u003cp\u003eThese findings support the critical role of PPG neuronal density in regulating cerebral vascular tone. Modulation of the PPG has also been employed in the treatment of conditions such as migraine, cluster headache, and trigeminal neuralgia [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Electrical stimulation of the SPG has been found to alleviate vasospasm in experimental models of SAH [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study provides valuable insights into potential therapeutic strategies for managing cerebral vasospasm following SAH. In particular, modulation of the PPG or its downstream neural pathways may represent a promising target for both prevention and treatment of cerebral vasospasm. The reduced parasympathetic nerve density observed in patients with Alzheimer\u0026rsquo;s disease may also contribute to the development of cerebrovascular disorders [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. SPG stimulation has been shown to reduce vasospasm by increasing the diameter of arteries within the circle of Willis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additional experimental studies suggest that SPG stimulation may have further beneficial effects following SAH. Yarnitsky et al. reported that SPG stimulation increased cerebral blood flow (CBF), reduced infarct size, and improved neurological function following permanent middle cerebral artery (MCA) occlusion. Henninger and Fisher demonstrated a reduction in infarct volume associated with improved perfusion in penumbral regions. Other neuromodulatory approaches, such as stimulation of the fastigial nucleus, have been shown to reduce neurogenic inflammation, spreading depression, and neuronal apoptosis, offering protection following cerebral ischemia [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLimitations and Future Directions\u003c/h2\u003e \u003cp\u003eThis study has several limitations. The experimental model may not fully replicate human physiology, and the sample size was relatively small. Future studies should aim to validate these findings using larger sample sizes and various animal models. In addition, advanced translational research is needed to assess the clinical efficacy of PPG modulation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study elucidates the complex relationship between APA vasospasm and PPG neuronal density, contributing to the development of novel approaches for the prevention and treatment of cerebral vasospasm.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthical Approval\u003c/h2\u003e \u003cp\u003e This study was approved by the Clinical Research Ethics Committee of Atat\u0026uuml;rk University (Approval No: 2200369130, Date: 09.11.2022). All animal care and experimental procedures were conducted in accordance with institutional guidelines approved by the same committee.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompliance with guidelines:\u003c/h2\u003e \u003cp\u003eAll procedures performed in this study involving animals complied with national and international guidelines, including the Basel Declaration and the principles outlined by Animal Research Tomorrow (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://animalresearchtomorrow.org/en\u003c/span\u003e\u003cspan address=\"https://animalresearchtomorrow.org/en\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.E. Y.C. U.T and C.K. wrote the main text of the manuscript, contributed to the experimental part of the study, and performed the statistical calculations. A.T and E.D. prepared the pathological samples and created figures 1-8. All authors contributed to the study design, data analysis, and manuscript revision, and reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to express their sincere gratitude to Prof. Dr. Dumlu Aydın from the Department of Neurosurgery, Atat\u0026uuml;rk University Faculty of Medicine, for his valuable guidance in obtaining ethical approval and for his insightful contributions to the interpretation and evaluation of the study findings.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBederson, J.B., et al., \u003cem\u003eGuidelines for the management of aneurysmal subarachnoid hemorrhage: a statement for healthcare professionals from a special writing group of the Stroke Council\u003c/em\u003e, \u003cem\u003eAmerican Heart Association.\u003c/em\u003e Stroke, 2009. 40(3): p. 994\u0026ndash;1025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRhoton, A.L., Jr., K. Fujii, and B. Fradd, \u003cem\u003eMicrosurgical anatomy of the anterior choroidal artery\u003c/em\u003e. Surg Neurol, 1979. 12(2): p. 171\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHara, H., et al., \u003cem\u003eParasympathetic cerebrovascular innervation: an anterograde tracing from the sphenopalatine ganglion in the rat.\u003c/em\u003e Neurosurgery, 1993. 32(5): p. 822-7; discussion 827.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJakobsen, M., \u003cem\u003eRole of initial brain ischemia in subarachnoid hemorrhage following aneurysm rupture. A pathophysiological survey\u003c/em\u003e. Acta Neurol Scand Suppl, 1992. 141: p. 1\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKovacic, S., G. Bunc, and J. Ravnik, \u003cem\u003eCorrespondence between the time course of cerebral vasospasm and the level of cerebral dopamine-beta-hydroxylase in rabbits\u003c/em\u003e. Auton Neurosci, 2006. 130(1\u0026ndash;2): p. 28\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAydin, M.D., et al., \u003cem\u003eThe role of neuron numbers of the petrosal ganglion in the determination of blood pressure: an experimental study\u003c/em\u003e. Minim Invasive Neurosurg, 2006. 49(6): p. 359\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBleys, R.L., et al., \u003cem\u003ePerivascular nerves of the human basal cerebral arteries: II. Changes in aging and Alzheimer's disease\u003c/em\u003e. J Cereb Blood Flow Metab, 1996. 16(5): p. 1048\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToda, N., et al., \u003cem\u003ePreganglionic and postganglionic neurons responsible for cerebral vasodilation mediated by nitric oxide in anesthetized dogs\u003c/em\u003e. J Cereb Blood Flow Metab, 2000. 20(4): p. 700\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGundersen, H.J., et al., \u003cem\u003eSome new, simple and efficient stereological methods and their use in pathological research and diagnosis\u003c/em\u003e. Apmis, 1988. 96(5): p. 379\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHara, H., et al., \u003cem\u003eAcetylcholine and vasoactive intestinal peptide in cerebral blood vessels: effect of extirpation of the sphenopalatine ganglion\u003c/em\u003e. J Cereb Blood Flow Metab, 1989. 9(2): p. 204\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToda, N., et al., \u003cem\u003eCerebral vasodilatation induced by stimulation of the pterygopalatine ganglion and greater petrosal nerve in anesthetized monkeys\u003c/em\u003e. Neuroscience, 2000. 96(2): p. 393\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdvinsson, L., et al., \u003cem\u003eVIP (vasoactive intestinal polypeptide)-containing nerves of intracranial arteries in mammals\u003c/em\u003e. Cell Tissue Res, 1980. 208(1): p. 135\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdvinsson, L. and D.N. Krause, \u003cem\u003eCerebral blood flow and metabolism\u003c/em\u003e. 2002: Lippincott Williams \u0026amp; Wilkins.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYarnitsky, D., et al., \u003cem\u003eReversal of cerebral vasospasm by sphenopalatine ganglion stimulation in a dog model of subarachnoid hemorrhage\u003c/em\u003e. Surg Neurol, 2005. 64(1): p. 5\u0026ndash;11; discussion 11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoysen, N.C., D.N. Dragon, and W.T. Talman, \u003cem\u003eParasympathetic tonic dilatory influences on cerebral vessels\u003c/em\u003e. Auton Neurosci, 2009. 147(1\u0026ndash;2): p. 101\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakahashi, M., Z.D. Zhang, and R.L. Macdonald, \u003cem\u003eSphenopalatine ganglion stimulation for vasospasm after experimental subarachnoid hemorrhage\u003c/em\u003e. J Neurosurg, 2011. 114(4): p. 1104\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHo, K.W.D., R. Przkora, and S. Kumar, \u003cem\u003eSphenopalatine ganglion: block, radiofrequency ablation and neurostimulation - a systematic review\u003c/em\u003e. J Headache Pain, 2017. 18(1): p. 118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalea, E., et al., \u003cem\u003eStimulation of cerebellar fastigial nucleus inhibits interleukin-1beta-induced cerebrovascular inflammation\u003c/em\u003e. Am J Physiol, 1998. 275(6): p. H2053-63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReis, D.J., et al., \u003cem\u003eBrief electrical stimulation of cerebellar fastigial nucleus conditions long-lasting salvage from focal cerebral ischemia: conditioned central neurogenic neuroprotection\u003c/em\u003e. Brain Res, 1998. 780(1): p. 161\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pterygopalatine ganglion, anterior perforating arteries, subarachnoid hemorrhage, vasospasm","lastPublishedDoi":"10.21203/rs.3.rs-6253817/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6253817/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnterior perforating arteries (APA) play a critical role in cerebral circulation. Whether there is a correlation between APA vasospasm and the neuronal density of the pterygopalatine ganglion during subarachnoid hemorrhage (SAH) has not yet been elucidated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study included 21 rabbits, divided into three groups: control (n=5), sham (n=5), and SAH (n=11). SAH was induced by injecting autologous blood into the cisterna magna. Seven days post-induction, the animals were decapitated, and both the APA and pterygopalatine ganglia were stereologically analyzed. Vasospasm severity was quantified using the vasospasm index (VSI). VSI values and neuronal densities were statistically evaluated using the Kruskal-Wallis and Mann-Whitney U tests, with significance set at p \u0026lt; 0.005.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant differences were found among the groups in both VSI values and neuronal densities. The control group exhibited a VSI of 0.234 ± 0.031, the sham group 0.995 ± 0.121, and the SAH group showed 1.127 ± 0.654 in cases of mild vasospasm and 2.126 ± 0.986 in cases of severe vasospasm. Neuronal density was also significantly reduced in the SAH group compared to the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe neuronal density of the pterygopalatine ganglion may significantly influence the severity of APA vasospasm following subarachnoid hemorrhage.\u003c/p\u003e","manuscriptTitle":"The Role of Pterygopalatine Ganglion Neuron Density in the Severity of APA Vasospasm Following Subarachnoid Hemorrhage: A Preliminary Experimental Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 06:35:10","doi":"10.21203/rs.3.rs-6253817/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b8b52b3e-d96e-4d4c-8c3d-1a3f1b9de2e4","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-11T17:08:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-23 06:35:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6253817","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6253817","identity":"rs-6253817","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}