J.Neurol.Sci.[Turk]

Journal of Neurological Sciences [Turkish] 33:(4)# 56; 566-572, 2016 http://www.jns.dergisi.org/text.php3?id=1011

Research Article

Ultrastructural Changes in the Circumventricular Organs After Experimental Traumatic Brain Injury

Nurullah EDEBALİ1, Deniz BİLLUR2, Belgin CAN2, Bektas ACIKGOZ1 1Bülent Ecevit University, Faculty of Medicine, Neurosurgery, Zonguldak, Turkey 2Ankara

University, Faculty of Medicine, Histology and Embryology, Ankara, Turkey

Summary

Objectives: Trauma and bleeding can affect the functions of circumventricular organs (CVOs), which are essential for most autonomic and endocrine functions. The aim of this study was to investigate the ultrastructural changes at circumventricular organs after traumatic brain injury. Methods: Two experimental groups were used: Traumatic injury group and a control group, six Wistar albino rats each. Under general anaesthesia, a lead object weighing 450 g was allowed to fall freely from a height of 90 cm through a copper tube onto the metal discover the skulls of the rats. 48 hours after trauma and control animals were anaesthetized. Rats were perfused with a fixation fluid (3 % paraformaldehyde and 1 % gluteraldehyde in PBS) until the heart stopped. Tissues containing SFO, OVLT, ME, AP were obtained using Paxinos & Watson Rat Stereotaxic Atlas. Ultrastructure examinations were done using TEM. Results: In all traumatic animals, edematous areas were abundant in neuropil. There were degeneration findings at neural and glial cell bodies, neuropil and myelinated nerve fibres. Degeneration at myelin sheath and degenerated organel remnants within the axoplasm were seen. There were disruption at myelinated and nonmyelinated axons. The number of the secretory granules at ME were reduced. Discussion: Changes observed in CVOs 48 hours after experimental head injury has a potential role in explaining functional and endocrine dysfunctions seen after TBI.

Key words: Circumventricular organs, traumatic brain injury, transmission electron microscopy

Deneysel Kafa Travması Sonrası Sirkümventriküler Organlardaki Ultrastrüktürel Değişiklikler

Özet Amaç: Beyinde otonomik ve endokrin fonksiyonları olan sirkümventriküler organlar travma ve/veya kanama sonucu etkilenmektedir. Bu çalışmanın amacı deneysel kafa travması sonrası sirkümventriküler organları ultrastrüktürel olarak incelemektir. Metod: Çalışma 6 deney hayvanından (Wistar albino sıçanlar) oluşan kontrol ve travma grubu olarak düzenlenmiştir. Deney grubunda genel anestezi altında sıçanların kafatasına bir tüp içerisinde 90 cm yükseklikten 450 gramlık kurşun ağırlık düşürülmüştür. Kontrol sıçanlar ve 48 saat sonunda deney hayvanları uyutularak %3 paraformaldehit ve %1gluteraldehit içeren PBS ile kalp durana dek perfüze edilmişlerdir. Sirkümventriküler organlar ( SFO, OVLT, ME, AP) Paxinos&Watson Stereotaksik sıçan atlası referanslarına göre çıkarılmıştır. Ultrastrüktür çalışmaları TEM ile yapılmıştır. Sonuç: Tüm travma dokularında nöropilde ödem saptanmıştır. Nöral ve gliyal hücrelerde, nöropil ve miyelinli sinir liflerinde dejenerasyon bulguları mevcuttur. Miyelin kılıflarında

566

J.Neurol.Sci.[Turk]

dejenerasyon ve aksoplazmada dejenere organel katlantıları saptanmıştır. Miyelinli ve miyelinsiz aksonlarda kırılmalar mevcuttur. ME"de sekretuvar granül sayıları azalmıştır. Tartışma: Bu çalışmada deneysel kafa travması sonrası sirkümventriküler organlarda 48. saatte saptanan bulgular klinikte görülen fonksiyonel ve endokrin bozuklukların açıklanmasına ışık tutabilir.

Anahtar Kelimeler: Sirkümventriküler organlar, travmatik beyin hasarı, transmisyon elektron mikroskobisi

INTRODUCTION Traumatic brain injury (TBI) is a pathological condition which may cause severe mortality and morbidity(2,9,10). After traumatic brain injury besides the cortical areas nearby the injury site, deep brain structures such as neural, glial and vascular tissues may be influenced. These deep structures may be injured primarily due to the impact but mostly by secondary mechanisms such as biochemical and immunological changes causing neural or glial cell dysfunction(9,4,12,21,24). In previous studies after experimental traumatic brain injury the changes in neural, glial and vascular tissues by electron microscopy were documented(8,16,17,22).

Sensory circumventricular organs (CVOs) such as, area postrema (AP), organum vasculosum lamina terminalis (OVLT) and subfornicial organ (SFO) and a secretory circumventricular organ, median eminence (ME), are located periventricular particularly around the third ventricle. They are neurotransmitter rich structures that are devoid of a blood-brain barrier and are known for their secretory role controlling fluid and electrolyte balance, thirst, reproduction(3,11), food intake(20), cardiovascular regulation and in the generation of central acute immune and febrile responses(11).

Although the role of CVO’s in physiological events have been well documented, there appears a lack of information in pathological conditions such as head injury. Our review of the literature failed to find reports specifically investigating

circumventricular organs by electron microscopy after experimental TBI. The aim of this study is to investigate the ultrastructural morphology at circumventricular organs SFO, OVLT, ME and AP 48 hours after cerebral contusion induced by a modified weight drop method(14) in rats.

MATERIAL AND METHODS The study protocol was approved by the Bülent Ecevit University Animal Ethics committee. The experiments were performed at the Experimental Surgery, Research and Animal Laboratory of Bülent Ecevit University, Faculty of Medicine, Zonguldak, Turkey. Ultrastructural examinations were performed at Ankara University, Faculty of Medicine, Department of Histology and Embryology, Ankara, Turkey. Wistar albino adult male rats, weighing 350-400 g on average were used in this study. The rats were divided into trauma and control groups. Each group consisted of six rats.

Surgical procedure A moderate brain-injury model, described by Marmarou et al.(14) and modified by Uçar et al.(23) was applied for head trauma to become a closed one and lead to reproducible brain injury. Anaesthesia under spontaneous respiration was enabled with intraperitoneal administration of 50 mg/kg body weight ketamine hydrochloride (Ketalar, 50 mg/ml, 10 ml vial, Pfizer İlaçları Ltd. Istanbul) and 10 mg/kg body weight xylazine hydrochloride (Rompun, 2% solution, 50 cc vial, Bayer-Türk İlaç Ltd., Istanbul). The rats were

567

J.Neurol.Sci.[Turk]

placed in a prone position on the table. A midline incision was made on the head, between the coronal and lambdoid sutures. A metallic disc of 10-mm diameter and 3-mm thickness was fixed to the cranium using bone wax between the two sutures and in the midline. Trauma was applied at the point where the metal disc was placed in the midline. A lead object weighing 450 g was allowed to fall freely from a height of 90 cm through a copper tube onto the metal disc over the skulls of the rats. The applied energy was calculated as: E=m.g.h m(mass) =450 g (0.450 kg), g (gravitational constant)= 9.8 m/s², h (height )=90 cm.(0.90 m.) E= 0.450 x 9.8 x 0.90 E= 4.9690 Joules.

Forty eight hours after, control and trauma animals were anaesthetized. After thoracotomy, the left ventricle was cannulated, the descending aorta clamped and the blood washed from the head and cervical regions with Ringer lactate solution after section of the vena cava.The bloodstream was then perfused with a fixation fluid (3 % paraformaldehyde and 1 % gluteraldehyde in PBS) until the heart stopped. After fixation in situ for 1 hour the objects were decapitated and specimens from CVO’s were obtained according to Paxinos & Watson Rat Streotaxic Atlas(3).

Electron microscopy investigation For the transmission electron microscope (TEM) analyses, samples were fixed with phosphate buffered (pH 7.3) 2.5% glutaraldehyde and a 2% PFA mixture solution for 2-4 h at room temperature. They were washed with phosphate-buffered saline solution (PBS, pH 7.3) and fixed with 1% osmium tetraoxide for 2 hours as the secondary fixative. After washing, they were embedded in Araldite 6005 and cut with a Leica Ultracut R (Leica, Solms, Germany) ultramicrotome. One micrometer semi-thin sections were stained by toluidine blue-Azur II to select the region of interest for the following

procedures. 60-70 nm thin sections were stained with uranil acetate and lead citrate. The sections were examined and photographed using a LEO 906 E TEM (80 kV, Oberkochen, Germany) microscope.

RESULTS Control Group In all samples of the control groups, neural and glial cell bodies, neuropil (interneural areas) and myelinated nerve fibers were found in normal appearance (Fig 1). Especially at ME, the cytoplasm of some neurons were filled with secretory granules (Fig 2). The capillary lumen and endothelial cells around them were in normal appearance.

Trauma Group In all animals, edematous areas were abundant in neuropil. There were degeneration findings at neural and glial cell bodies, neuropil and myelinated nerve fibres (Fig 3,4). There were narrowing of the capillary lumen and degenerative changes at the cytoplasm of glial cell process lying near the capillary wall. Perivascular edema was found (Fig 3). In some oligodendrocytes, there were swelling as well as degeneration of the mitochondria, dilatation and lack of rough endoplasmic reticulum (Fig 4). Depletion of secretory granules in neurons was observed especially in ME. The cell was shurunked and degenerated, the cytoplasm was filled with vacuoles and degenerated mitochondria. The nucleus was contracted, chromatin material looked like dense masses and its nucleolus was located at peripherially of the nucleus (Fig 5). Mitochondrial degeneration, increased number of lysosomes were observed in most of the neural cytoplasma. There were axonal disruption at myelinated and nonmyelinated axons. Degeneration at myelin sheath, degenerated organel remnants within the axoplasma were also observed (Fig 6).

568

J.Neurol.Sci.[Turk]

Figure 1: Electron micrograph of SFO from control group showing glial cell process (arrow head) near the capillary lumen (c), myelinated nerve fibers (arrow) and neuropil (n) with normal appearance. Uranyl acetate/lead citrate x 12930.

Figure 2: Electron micrograph of ME, a normal neural cell architecture from an animal in control group. A lot of secretory granule (arrow), abundant rough endoplasmic reticulum (tailed arrow), a few mitochondria and a large nucleus (N) is seen. n; neuropil. Uranyl acetate/lead citrate x 7750.

Figure 3: Electron micrograph of ME from 48 hours after TBI. Showing abundant edematous areas (star) in neuropil (n)and araund the capillaries (c). The neuron is shrunken. The nucleus (N) is contracted and the cytoplasm is decreased. Uranyl acetate/lead citrate x 3597.

Figure 4: Electron micrograph of SFO from 48 hours after TBI, where an altered glial cell (Gli) (probably oligodendroglia) showing a swollen and degenerated mitochondria (m), rough endoplasmic reticulum (RER) dilatation and lack of RER is seen. The nucleus presents a normal aspect. In the neuropil the degenerative changes at myelin sheath (arrow) can be noted. Tailed arrow: RER dilatation Uranyl acetate/lead citrate x 10000.

569

J.Neurol.Sci.[Turk]

DISCUSSION In this study forty-eight hours after TBI we found changes within the neurons and glial cells of the studied circumventricular organs SFO, OVLT, AP and ME. Edema was observed in the cells. The findings such as swollen mitochondria, dilatation at rough endoplasmic reticulum and increased number of lysosomes were the result of the trauma.

Dietrich et al, in their electrıon microscopy study in rat brain tissues, found similar findings(8). According to the authors, the presence of "pyknotic nuclei" demonstrated the severity of the injury(8). In our study we did not observe pyknotic nuclei.

In several types of experimental head injury studies such as fluid percussion model(8,16), pneumatic model(22), cortical, intraparenchymal, subarachnoidal and

ventricular hemorrhages were noted. In our study subarachnoid and intraventricular hemorrhage (Frank hemorrhage) was not noted, we believe, factors other than blood borne degradation products played role in the morphological changes.

The presence of axonal ruptures seen in some cases together with the above mentioned changes within the cells are important from clinical point of view. The neurons of sensory circumventricular organs such as SFO, OVLT and AP have a rich network of neural connection between the brain centers. The OVLT, SFO and AP are integrated to the hypothalamus and the dorsal vagal complex(19). SFO and OVLT mainly take part in the fluid and electrolyte balance, food intake and febril responses. The abnormalities seen in clinical studies, especially in patients with diffuse axonal injury, may potentially occur due to the disturbed morphology and function of

Figure 5: Electron micrograph of ME from 48 hours after TBI showing a degenerated neuron (N). Notice the mitochondrial degeneration (m), depletion of secretory granules (arrow head) and peripheral located nucleolus (nu). Disorganization of the myelin (arrow) is seen in some myelinated fibers. Uranyl acetate/lead citrate x 7750.

Figure 6: Electron micrograph of SFO from 48 hours after TBI. Degenerated mitochondria (m) of neuron (N) cytoplasm, lysosomes (L) can be seen. Degenerative changes of myelin sheath (bold arrow), axonal disruption of unmyelinated axon (spiral arrow) can be seen. Glial cell (Gli) nearby capillary (c) and synaptic vesicles (sv) are seen. En; endothelial cell nucleus, arrow; myelinated nerve fiber. Asterix: degenerated organel remnants in axoplasma. Uranyl acetate/lead citrate x 7750.

570

J.Neurol.Sci.[Turk]

CVO’s. Similarly cardiac abormalities, rythm problems and even sudden myocardial dysfunction seen after TBI may have a possible relation with the affected AP.

Abnormal secretion of pituitary hormones were well described after TBI. Although depletion of neurosecretory granules may occur in physiologic conditions, generally in pathological conditions because of decreased synthesis, occupation of the granules by abnormal proteins, and increased secretion rate due to an impulse may decrease the number of secretory granules(13,7). We have observed the depletion of secretory granules in the trauma group, in the ME (the only secretory CVO we have studied). After TBI the disturbed blood brain barrier was demonstraded in previous studies(1). Disruption of the blood brain barrier after TBI is important because it leads to the pass of blood borne factors, such as vasoactive serotonin, tromboxane and excitotoxic glutamate, quinolic acid from the barrier and increases the effects of the trauma(8).

Although CVO’s remain therotically outside the blood brain barrier, previous studies had shown that pericapillary areas and outer external laminae has some properties serving as a barrier to avoid free passage from the blood and CSF(19,6). In our study in the trauma group narrowing of the pericapillary areas, broken and seperated outer external laminae, had given the impression of disturbance of the above mentioned barrier.

CONCLUSION The ultrastructure changes observed in the present study has to be evaluated in a time course manner in the future. Recently, the presence of neural stem cells has been described in CVOs in both human autopsy studies(18,15) and in animal studies(5). The presence of neural stem cells in the CVOs is important for future investigations especially in the healing of severely

injured people. Future research must also address both the restoration of changes due to primary insult after TBI and to the changes due to secondary mechanisms. Conflict of Interest: The authors declare no conflict of interest.

Correspondence to: Nurullah Edebali E-mail: nedeb@superonline.com Received by: 24 November 2015 Accepted: 20 August 2016 The Online Journal of Neurological Sciences (Turkish) 1984-2016 This e-journal is run by Ege University Faculty of Medicine, Dept. of Neurological Surgery, Bornova, Izmir-35100TR as part of the Ege Neurological Surgery World Wide Web service. Comments and feedback: E-mail: editor@jns.dergisi.org URL: http://www.jns.dergisi.org Journal of Neurological Sciences (Turkish) Abbr: J. Neurol. Sci.[Turk] ISSNe 1302-1664 REFERENCES

1. Abbott NJ, Patabendige AAK, Dolman DEM, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiology of Disease. 2010;37:13-25.

2. Adams JH, Jennett B, McLellan DR, Murray LS, Graham DI. The neuropathology of the vegetative state after head injury. J Clin Pathol. 1999; 52: 804-6.

3. Akpinar G, Açikgöz B, Sürücü S, Celik HH, Cağavi F. Ultrastructural changes in the circumventricular organs after experimental subarachnoid hemorrhage. Neurol Res.2005;27:580-5.

571

J.Neurol.Sci.[Turk]

4. Ates O, Caylı S, Altınoz E, Gurses I, Yucel N, Sener M et al. Neuroprotection by resveratrol against traumatic brain injury in rats. Moll Cell Biochem. 2007; 294:137-44.

5. Bennett L, Yang M, Enikolopov G, Iacovitti L. Circumventricular organs: A novel site of neural stem cells in the adult brain. Mol Cell Neurosci. 2009;41: 337-47.

6. Bouchaud C, Le Bert M, Dupouey P. Are close contacts between astrocytes and endothelial cells a prerequisite condition of a blood-brain barrier? The rat subfornical organ as an example. Bio Cell.1989;67:159-65.

7. Burgoyne RD, Morgan A.Secretory granule exocytosis. Physiol Rev .2003; 83: 581-632.

8. Dietrich WD, AlonsoO, Halley M.Early microvascular and neuronal consequences of traumatic brain injury: A light and electronmicroscopic study in rats.J Neurotrauma. 1994; 11: 289-301.

9. Di Leonardi AM, Huh JW, Raghupathi R. Impaired axonal transport and neurofilament compaction ocur in seperate populations of injured axons following diffuse brain injury in the immature rat. Brain Res.2009; 1263: 174-82.

10. Gennarelli TA, Thibault LE, Adams JH, Graham DI, Thompson CJ, Marcincin RP. Diffuse axonal injury and traumatic coma in the primate. Ann Neurol.1982;12:564-74.

11. Gross PM, Weindl A. Peering through the windows of the brain.Review. J Cereb Blood Flow Metab.1987;7:663-72.

12. KalaycıM, Unal MM, Gul S, Acikgoz S, Kandemir N, Hanci V, Edebali N, Acikgoz B. Effect of coenzyme Q10 on ischemia and neuronal damage in an experimental traumatic brain-injury model in rats.BMC Neuroscience.2011;12:75.

13. Kelly P, Feakins R, Domizio P, Murphy J, Bevins C, Wilson J etal.Paneth cell granule depletion in the human small intestine under infective and nutritional stress. Clin Exp Immunol.2004; 135:303-9.

14. Marmarou A, Foda MA, Van den Brink W, Campbell J, Kita H, Demetriadou K. A new model of diffuse brain injury in rats.Part I: Pathophysiology and biomechanics. J Neurosurg.1994;80:291-300.

15. Nogueira AB, Sogayar MC, Colquhoun A, Siqueira SA, Nogueira AB, Marchiori PE et al. Existence of a potential neurogenic system in the adult human brain. J Transl Med. 2014; 12:75.

16. Pettus EH, Christman CW, Giebel ML, Povlishock JT. Traumatically induced altered membrane permeability: Its relationship to traumatically induced reactive axonal change. J Neurotrauma.1994;11:507-22.

17. Pettus EH, Povlishock JT.Characterization of a distinct set of intra-axonal ultrastructural cahanges associated with traumatically induced alteration in axolemnal permeability. Brain Res. 1996; 722:1-11.

18. Sanin V, Heeß C, Kretzschmar HA, Schüller U.Recruitment of neural precursor cells from circumventricular organs of patients with

cerebral ischaemia. Neuropathol Appl Neurobiol.2013;39: 510-18.

19. Sisó S, Jeffrey M, González L. Sensory circumventricular organs in health and disease.Acta Neuropathol. 2010;120:689-705.

20. Smith PM, Ferguson AV. Circulating signals as critical regulators of autonomic state - central roles for the subfornical organ.Am J Physiol Regul Integr Comp Physiol. 2010; 299: R405?15.

21. Soares HD, Hicks RR, Smith D, McIntosh TK. Inflammatory leukocytic recruiment and diffuse neuronal degeneration are seperate pathological processes resulting from traumatic brain injury. J Neurosci.1995;15:8223-33.

22. Sutton RL, Lescaudron L, Stein DG. Unilateral cortical contusion injury in the rat: Vascular disruption and temporal development of cortical necrosis. J Neurotrauma.1993;10:135-49.

23. Ucar T, Tanriover G, Gurer I, Onal MZ, Kazan S. Modified experimental mild traumatic brain injury model. J Trauma.2006;60:558-65.

24. Uhl MW, Biagas KV, Grundl PD, Barmada MA, Schiding JK, Nemoto EM et al. Effects of neutropenia on edema, histology, and cerebral blood flow after traumatic brain injury in rats. J Neurotrauma.1994;11:303-15.

572