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Cervical sympathectomy modulates the neurogenic inflammatory neuropeptides following experimental subarachnoid hemorrhage in rats
Brain Research 1722 (2019) 146366
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Research report
Cervical sympathectomy modulates the neurogenic inflammatory neuropeptides following experimental subarachnoid hemorrhage in rats☆
T
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Yasar Dagistana, Erkan Kilincb, , Cemre Nur Balcic a
Abant Izzet Baysal University, Medical Faculty, Department of Neurosurgery, Bolu, Turkey Abant Izzet Baysal University, Medical Faculty, Department of Physiology, Bolu, Turkey c Abant Izzet Baysal University, Institute of Health Sciences, Department of Histology and Embryology, Bolu, Turkey b
H I GH L IG H T S
hemorrhage induces c-fos expression in the trigeminal nucleus caudalis. • Subarachnoid attenuates subarachnoid hemorrhage-induced c-fos expression. • Sympathectomy hemorrhage induces release of neurogenic inflammatory neuropeptides. • Subarachnoid • Sympathectomy modulates induced release of neurogenic inflammatory neuropeptides.
A R T I C LE I N FO
A B S T R A C T
Keywords: Subarachnoid hemorrhage Cervical sympathectomy Neuropeptides Neurogenic inflammation Trigeminovascular system
Background: Neuroinflammation is implicated in cerebral vasospasm and brain injuries after subarachnoid hemorrhage (SAH). In addition to classical neuroinflammation with increased inflammatory cytokines, a sterile neurogenic inflammation characterized by release of potent vasoactive neuropeptides may be responsible for brain injuries after SAH. Sympathetic discharges from superior cervical ganglion contribute to vasoconstriction of cerebral arteries Thus, we investigated the effects of surgical cervical sympathectomy on the neurogenic inflammatory neuropeptides shortly after SAH induction in a model of SAH in rats. Methods: Male Wistar rats were divided into 4 groups: control; was not touched, saline group; 300 μl of saline was injected into prechiasmatic cistern, SAH+Sham group; 300 μl of autologous blood was injected to induce subarachnoid hemorrhage into prechiasmatic cistern; SAH+Symp group; the left cervical sympathetic branch was surgically removed after the induction of SAH. Levels of neuropeptides CGRP, SP and VIP which are responsible for neurogenic inflammation, in plasma, trigeminal ganglion, brainstem and brain tissue were measured by ELISA. In addition, c-fos expression as a marker of neuronal activation in the trigeminal nucleus caudalis (TNC) was determined by immunohistochemical staining. Results: SAH significantly increased c-fos expression in the TNC, as well as CGRP, SP and VIP concentrations in plasma and trigeminal ganglion neurons, and also CGRP and SP concentrations in the brainstem. Cervical sympathectomy application significantly reduced the increases in these parameters induced by SAH. Conclusions: Our findings suggest that cervical sympathectomy treatment may prevent early brain injury by modulating SAH-induced neurogenic inflammatory neuropeptides such as CGRP, SP and VIP, and improve the quality of life in survivors following SAH.
1. Introduction Subarachnoid hemorrhage (SAH) is a life-threatening neurological problem with 50% of pre-hospital deaths (Campos-Pires et al., 2016). The most common cause of SAH, except for trauma is the rupture of an
aneurysm in the brain (Goksu et al., 2016). It has been reported that approximately 75% of patients with aneurysmal SAH die and the most survivors are severely disabled (Hop et al., 1997). Sudden and severe headache is the most common warning symptom of SAH and it chronically persists in the survivors after SAH (Ferro and Melo, 1998;
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Presentation at a conference: This study has been presented as oral presentation at 15th Pain Congress with International Participation on November 15 – 18, 2018, in Antalya, Turkey. ⁎ Corresponding author. E-mail address: [email protected] (E. Kilinc). https://doi.org/10.1016/j.brainres.2019.146366 Received 9 March 2019; Received in revised form 6 August 2019; Accepted 7 August 2019 Available online 08 August 2019 0006-8993/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Neurological scores after subarachnoid hemorrhage. (A) Neurological score of each group at 2th, 8th and 14th days post-SAH, (B) mean neurological score of groups at 2th, 8th and 14th days postSAH. **P < 0.01 (SAH + Sham vs Saline, and ##P < 0.01 (SAH + Symp vs SAH + Sham). SAH: subarachnoid hemorrhage; Symp: sympathectomy.
was reported that vasoconstriction in middle and basal cerebral arteries occured following induction of experimental SAH in rats, however, this vasospasm was reduced by cervical sympathectomy application (Hu et al., 2014). Therefore, in the present study, we aimed to investigate the effects of postoperative superior cervical sympathectomy on the cfos expression, a marker of neuronal activation, in the trigeminal nucleus caudalis and levels of neuropeptides (CGRP, SP and VIP) leading to neurogenic inflammation in the plasma and strategical structures (trigeminal ganglion, brainstem and brain tissue) related to their expression and release in a model of SAH in rats.
Chen and Ayata, 2016). It is well established that non-infectious neuroinflammation plays a key role in the pathophysiology of SAH (Zheng and Wong, 2017). Current data report that this neuroinflammation contributes to early brain injury, cerebral vasospasm and consequently delayed neurological deterioration and the disabilities after SAH (Zheng and Wong, 2017; Lucke-Wold et al., 2016; de Oliveira Manoel and Macdonald, 2018). In addition to classical neuroinflammation with increased inflammatory cytokines such as IL-1β, IL-6, TNF-α etc, a sterile neurogenic inflammation characterized by the release of potent vasoactive neuropeptides can occur following SAH. Vasoactive neuropeptides including calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP) are considered to be key mediators in neurogenic inflammation (Lewis et al., 2013; Corrigan et al., 2016; Messlinger et al., 2011; Schytz et al., 2010). It was suggested that neurogenic inflammatory response can exacerbate classical neuroinflammation via a positive feedback loop with classical inflammatory mediators (Corrigan et al., 2016). However, inflammation and oxidative stress mediators, and protons following SAH can induce both expression of vasoactive neuropeptides in trigeminal ganglion neurons and their release from peripheral and central terminals of trigeminal nerve fibers in the cranial dura mater and brainstem, respectively (Berczi et al., 2010; Ichikawa and Sugimoto, 2002; Raddant and Russo, 2014). Moreover, increased intracranial pressure due to SAH may also lead to release of these vasoactive neuropeptides by activating mechanoreceptors in trigeminal afferent nerve terminals innervating meninges due to these neuropeptides-expressing neurons are sensitive to noxious mechanical stimuli (Iyengar et al., 2017). Upon released, CGRP and VIP cause vasodilation of cerebral blood vessels, however, SP leads to extravasation of plasma proteins (Levy et al., 2018; Ramachandran, 2018). As a result of all of these events, a sterile neurogenic inflammation in the brain and meninges occurs. Interestingly, CGRP and SP released from trigeminal sensory nerve fibers innervating meningeal blood vessels further promote neurogenic inflammation by activating mast cells, which are resident immune cells in the meninges (Ottosson and Edvinsson, 1997; Ramachandran, 2018; Strassman et al., 1996; Theoharides et al., 2005). In addition, CGRP and SP also induce the initiation of headache due to neurogenic inflammation by sensitizing sensory nerve terminals and they facilitate transmission of pain signals to second-order neurons in trigeminal nucleus caudalis (TNC) in the brainstem (Strassman et al., 1996), where it harbours central terminals of trigeminal afferent nerve fibers. The superior cervical ganglion is a part of the sympathetic nervous system and supplies sympathetic innervation to the head and neck. It
2. Results 2.1. TCSG treatment ameliorated neurological function in rats with SAH It was not observed animal deaths in the sham group, but the mortality rate of rats in SAH group was bigger at 48 hr (6/20; 30%) than that in SAH + Symp group (4/18; 22.2%). The neurological scores at 2th days post-SAH for control group was 0 ± 0, for Saline group was 0.42 ± 0.2, for SAH + Sham group was 10.8 ± 0.59, and for SAH + Symp group was 5.8 ± 0.45. The neurological scores at 8th days post-SAH for control group was 0 ± 0, for Saline group was 0.28 ± 0.18, for SAH + Sham group was 11.5 ± 0.36, and for SAH + Symp group was 6.4 ± 0.52. The neurological scores at 14th days post-SAH for control group was 0 ± 0, for Saline group was 0.14 ± 0.1, for SAH + Sham group was 10.5 ± 0.57, and for SAH + Symp group was 5.7 ± 0.52. Mean values of the neurological scores at these 3 different set point for control group was 0 ± 0, for Saline group was 0.28 ± 0.15, for SAH + Sham group was 11 ± 0.34, and for SAH + Symp group was 6 ± 0.39. The neurological scores at 2th, 8th, and 14th days post-SAH in SAH + Sham group rats were higher compared to the Saline group, respectively (P = 0.004, P = 0.002 and P = 0.005, Fig. 1A). Following TCSG, these rats showed a significant reducing in motor and behavioral deficits compared to the SAH + Sham groups, respectively (SAH + Symp vs SAH + Sham, P = 0.009, P = 0.004 and P = 0.008, Fig. 1A). Likewise, in terms of mean values of the neurological scores at these 3 different set point, rats in SAH + Sham group showed higher the neurological scores than that in the Saline group (SAH + Sham vs Saline, P = 0.0021, Fig. 1B), and following TCSG, these rats exhibited a significant reducing in motor and behavioral deficits (SAH + Symp vs SAH + Sham, P = 0.0041, Fig. 1B). 2
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Fig. 2. Effects of subarachnoid hemorrhage induction and sympathectomy treatment on the c-fos expression in trigeminal nucleus caudalis in the brainstem. (A) c-fos positive neurons in trigeminal nucleus caudalis in control group, (B) in saline group, (C) in SAH + Sham group and (D) in SAH + Symp group, (E) while SAH increased the number of c-fos positive neurons, the sympathectomy treatment reduced these increases in trigeminal nucleus caudalis in the brainstem (the number of c-fos positive neurons is given as per section in the graph). Arrowheads demonstrate c-fos positive neurons in trigeminal nucleus caudalis. Please note, increased the number of c-fos positive neurons in the SAH + Sham group compared to saline, and decrease in SAH + Symp group compared to SAH + Sham group. ***P < 0.001 (SAH + Sham vs Saline), and #P < 0.05 (SAH + Symp vs SAH + Sham). SAH: subarachnoid hemorrhage; Symp: sympathectomy.
290.8 ± 19 pg/ml to 212.3 ± 18.4 (SAH + Symp vs SAH + Sham, P = 0.003, Fig. 4A) and VIP concentrations from 29.1 ± 2.9 pg/ml to 21.3 ± 1.2 (SAH + Symp vs SAH + Sham, P = 0.028, Fig. 5A) in the plasma respectively, compared to SAH.
2.2. TCSG reversed up-regulation of c-fos protein expression in TNC The increase in c-fos expression in TNC is a key marker of neuronal activation that indicates also the activation of trigeminovascular system (Bullitt, 1990). In addition, neurogenic inflammation plays a key role in activation and sensitization of the trigeminovascular system (Levy, 2012). Thus we explored c-fos expression in TNC in experimental SAH model in current study. In the present study, the number of c-fos positive neurons in the TNC were 13 ± 1.0 in per section in control group (Fig. 2A and E). However, SAH increased the number of c-fos positive neurons in the TNC from 15 ± 1.0 to 43 ± 1.9 in per section compared to the Saline group (SAH + Sham vs Saline, P = 0.001, Fig. 2B, C and E), on the other hand, TCSG treatment in the SAH + Symp group reduced SAH-induced the increase in the number of c-fos positive neurons from 43 ± 1.9 to 34 ± 1.7 in per section compared to SAH + Sham group (P = 0.011, Fig. 2C, D and E).
2.5. TCSG reversed up-regulation of CGRP, SP and VIP concentrations in trigeminal ganglion SAH increased CGRP concentrations from 55.8 ± 3.3 pg/mg/ml to 73.9 ± 4.2 pg/mg/ml (SAH + Sham vs Saline, P = 0.004, Fig. 3B) and SP concentrations from 434.3 ± 60.4 pg/mg/ml to 786.6 ± 34.9 pg/ mg/ml (SAH + Sham vs Saline, P = 0.001, Fig. 4B) and VIP concentrations from 85.8 ± 4.9 pg/mg/ml to 145.8 ± 22.2 pg/mg/ml (SAH + Sham vs Saline, SAH + Sham vs Saline, P = 0.011, 5B) in trigeminal ganglion respectively, compared to Saline group. On the other hand, TCSG treatment decreased CGRP concentrations from 73.9 ± 4.2 pg/mg/ml to 60.3 ± 2.3 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.037, 3B) and SP concentrations from 786.6 ± 34.9 pg/mg/ml to 523.0 ± 51.8 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.005, 4B) and VIP concentrations from 145.8 ± 22.2 pg/mg/ml to 96.9 ± 9.2 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.047, 5B) in trigeminal ganglion respectively, compared to SAH + Sham group.
2.3. Effect of TCSG on vasoactive neuropeptides in SAH rats CGRP, SP and VIP are potent vasoactive neuropeptides that play key roles in the neurogenic inflammation (Lewis et al., 2013; Corrigan et al., 2016; Messlinger et al., 2011; Schytz et al., 2010). Therefore, in present study, we measured the concentrations of these vasoactive neuropeptides in plasma and in the structures related to their expression and release including trigeminal ganglion, brainstem and brain tissue.
2.6. TCSG reversed up-regulation of CGRP and SP concentrations in brainstem
2.4. TCSG reversed up-regulation of CGRP, SP and VIP concentrations in plasma
While SAH increased CGRP concentrations from 25.1 ± 1.7 pg/ mg/ml to 41.7 ± 4.1 pg/mg/ml (SAH + Sham vs Saline, P = 0.005, Fig. 3C) and SP concentrations from 321.1 ± 38.9 pg/mg/ml to 593.1 ± 73.4 pg/mg/ml (SAH + Sham vs Saline, P = 0.003, Fig. 4C), it did not change VIP concentrations (from 49.4 ± 3.4 pg/mg/ml to 56.7 ± 4.8 pg/mg/ml, SAH + Sham vs Saline, P = 0.65, Fig. 5C) in brainstem, respectively compared to saline group. On the other hand, TCSG treatment decreased CGRP concentrations from 41.7 ± 4.1 pg/ mg/ml to 29.8 ± 3.3 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.04, Fig. 3C) and SP concentrations from 593.1 ± 73.4 pg/mg/ml to 369.8 ± 36.7 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.016,
SAH increased CGRP concentrations from 24.6 ± 1.0 pg/ml to 37.1 ± 1.3 pg/ml (SAH + Sham vs Saline, P = 0.001, Fig. 3A) and SP concentrations from 151.9 ± 7.2 pg/ml to 290.8 ± 19 pg/ml (SAH + Sham vs Saline, P = 0.001, Fig. 4A) and VIP concentrations from 17.2 ± 1.3 pg/ml to 29.1 ± 2.9 pg/ml (SAH + Sham vs Saline, P = 0.001, Fig. 5A) in the plasma respectively, compared to Saline group. On the other hand, TCSG treatment decreased CGRP concentrations from 37.1 ± 1.3 pg/ml to 30.4 ± 0.6 pg/ml (SAH + Symp vs SAH + Sham, P = 0.001, Fig. 3A) and SP concentrations from 3
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Fig. 3. Effects of subarachnoid hemorrhage induction and sympathectomy treatment on CGRP concentrations in the plasma, trigeminal ganglion, brainstem and brain tissue. **P < 0.01, ***P < 0.001, #P < 0.05, ###P < 0.001. *: SAH + Sham vs Saline, #: SAH + Symp vs SAH + Sham.
brain injury after SAH (de Oliveira Manoel and Macdonald, 2018; Zheng and Wong, 2017; Lucke-Wold et al., 2016). 30% of patients, survivors following SAH, are exposed to delayed brain deterioration (Zheng and Wong, 2017). Delayed brain deterioration commonly shows up 3–14 days later and it is characterized by delayed cerebral ischemia and delayed neurological deficits (Chen et al., 2015). Cerebral vasospasm that appears in patients with SAH who survive following aneurysmal or non-aneurysmal SAH leads to the ischemic brain injury, and an increase in the level of pro-inflammatory cytokines and reactive oxygen species in the brain (Denes et al., 2010; Fujii et al., 2013). The resulting neroinflammation can cause the release of vasoactive and pro-nociceptive neuropeptides promoting the neurogenic inflammation such as CGRP, SP and VIP through axon reflex by stimulating receptors in trigeminal sensory nerve terminals, which are innervating the cranial dura mater (Levy et al., 2018; Lukács et al., 2015; Ramachandran, 2018). While CGRP and VIP cause the vasodilatation of meningeal blood vessels, SP gives rise to plasma protein extravasation by increasing the permeability of meningeal blood vessels (Levy et al., 2018; Ramachandran, 2018). Moreover, each of neuropeptides CGRP, SP and VIP also leads to the release of numerous inflammatory mediators from the mast cells in the cranial dura mater by degranulating them (Levy et al., 2018; Ottosson and Edvinsson, 1997; Theoharides et al., 2012). Mast cell activation would in turn further enhance dural neurogenic inflammation (Theoharides et al., 2012) As a domino,
Fig. 4C) in brainstem, respectively compared to SAH + Sham group. 2.7. SAH or TCSG did not change CGRP, SP and VIP concentrations in brain tissue Interestingly, neither induction of SAH nor TCSG treatment changed CGRP, SP and VIP concentrations in brain tissue. CGRP concentrations were 38.6 ± 3.0 pg/mg/ml in Saline group, 41.4 ± 4.5 pg/mg/ml SAH + Sham group, 40.0 ± 3.9 pg/mg/ml in SAH + Symp group; P = 0.93 for Saline group vs SAH + Sham (Fig. 3D), P = 0.99 for SAH + Sham versus SAH + Symp (Fig. 3D). SP concentrations were 270.3 ± 15.7 pg/mg/ml in Saline group, 287.6 ± 38.5 pg/mg/ml SAH + Sham group, 272.0 ± 20.9 pg/mg/ml in SAH + Symp group, P = 0.95 for Saline group vs SAH + Sham (Fig. 4D), P = 0.96 for SAH + Sham versus SAH + Symp (Fig. 4D). VIP concentrations were 116.0 ± 8.0 pg/mg/ml in Saline group, 127.4 ± 12.0 pg/mg/ml in SAH + Sham group, 118.9 ± 6.6 pg/mg/ml in SAH + Symp group, P = 0.83 for Saline group vs SAH + Sham group (Fig. 5D), P = 0.92 for SAH + Sham group vs SAH + Symp group(Fig. 5D). 3. Discussion Neuroinflammation following SAH contributes to early brain injury and then neuroinflammation processes participate in the secondary 4
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Fig. 4. Effects of subarachnoid hemorrhage induction and sympathectomy treatment on SP concentrations in the plasma, trigeminal ganglion, brainstem and brain tissue. **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01. *: SAH + Sham vs Saline, #: SAH + Symp vs SAH + Sham.
et al., 1998; Ostergaard, 1991; Schwedt and Matharu, 2006). Therefore, in our study, the sympathectomy treatment decreased SAH induced cfos expression suggesting that the sympathectomy treatment may alleviate the overactivation of trigeminovascular system and prevent chronic headache after SAH. The potent vasoactive neuropeptides CGRP, SP and VIP are closely associated with the generation of dural neurogenic inflammation (Schytz et al., 2010; Ramachandran, 2018). CGRP, SP and VIP are released upon activation of the trigeminal ganglion or fibers, and this release of the neuropeptides initiate a sterile neurogenic inflammation in the meninges (Messlinger et al., 1993; Moskowitz, 1993). Moreover this neurogenic inflammation also leads to the activation of mast cells in the cranial dura mater. Then dural mast cells further promote neurogenic inflammation by release of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, IFN-β (Theoharides et al., 2012; Conti et al., 2017; Caraffa et al., 2018) from their cytoplasmic granules. Therefore, it can be stated that CGRP, SP and VIP are chief triggers for dural neurogenic inflammation since they propagate neurogenic inflammation by activating immune mast cells in the meninges. Therefore in current study, we target the neurogenic inflammatory neuropeptides including CGRP, SP and VIP apart from the inflammatory cytokines in the classical inflammation. In the present study, we found that SAH increased plasma CGRP, SP and VIP concentrations. These findings are important due to the fact that increased plasma levels of these neuropeptides may be an indirect
neuroinflammation and cerebral vasospasm following SAH can evoke delayed brain deterioration, delayed cerebral ischemia, delayed neurological deficits and chronicity of headache after SAH. Therefore, in particular prevention of cerebral vasospasm after SAH may prevent neurogenic inflammation and by extension the other related impairments. In the direction of this hypothesis, in the present experimental study, we performed cervical sympathectomy following induction of SAH in the experimental model of SAH, and found that the sympathectomy reduced the increases in markers of neuronal activation and dural neurogenic inflammation including c-fos, CGRP, SP and VIP evoked by SAH. C-fos expression as a marker of neuronal activation in the brainstem is widely used to investigate neuronal activation based on disturbance of meninges in experimental studies (Kilinc et al., 2018; Zhang et al., 2017). In the present study, we found that c-fos expression was increased in TNC in SAH group. This finding is extremely important because it is well known that the trigeminovascular system is activated during head pain stemming from meningeal irritation by dural inflammation (Kilinc et al., 2017; Levy et al., 2018; Ramachandran, 2018). Thus, in our study, the increase in c-fos expression in SAH group indicates that SAH leads to the activation of the trigeminovascular system. The activation of the trigeminovascular system may be a sign of generation and transmission of head pain after SAH. Moreover, this finding is consistent with previous studies reporting that the rupture of a saccular intracranial aneurysm leads to thunderclap headache (Linn 5
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Fig. 5. Effects of subarachnoid hemorrhage induction and sympathectomy treatment on VIP concentrations in the plasma, trigeminal ganglion, brainstem and brain tissue. *P < 0.05, **P < 0.01, #P < 0.05. *: SAH + Sham vs Saline, #: SAH + Symp vs SAH + Sham.
possible reason for this may be that our investigation is a longer-term study covering 14 days. Because the symptoms of vasospasm based on SAH develop slowly throughout the time. In addition, in the present study, SAH increased the levels of CGRP, SP and VIP also in trigeminal ganglion neurons and the levels of CGRP and SP in the brainstem which are key structures indicating a painful inflammation in the brain and meninges. These findings are also in accord with the increase in the c-fos expression in TNC in the brainstem in the current study because of up-regulation in c-fos expression indicates transmission of pain signals from the head with neuroinflammation due to activation of the trigeminovascular system (Levy, 2012; Bullitt, 1990). Our these findings are important due to the fact that neuropeptides CGRP, SP and VIP released from meningeal nerve terminals are mostly expressed in the soma of trigeminal ganglion neurons (Goto et al., 2017; Lazarov, 2002). Moreover these findings suggest that SAH may evoke neurogenic inflammation through also activation of trigeminal ganglion neurons. In addition, SAH increased CGRP and SP levels without changing VIP levels in the brainstem. In this study, the increase in CGRP and SP levels in the brainstem indicates most probably a painful neuroinflammation of the brain and meninges. Moreover, SAH may lead to neurogenic inflammation not only in the meninges and brain but also in the brainstem via an increase CGRP and SP levels in the brainstem. On the other hand, SAH did not change VIP levels in the brainstem. This may be due to VIP expression in TNC is relatively less compared to those in trigeminal ganglion neurons.
reflection of neuroinflammation in the nervous system. Because, it was previously demonstrated that trigeminal ganglion, which is the major source of these neuropeptides, is outside of the blood–brain barrier (BBB) (Eftekhari et al., 2015). Moreover it was reported that SP can cross BBB through a carrier-mediated mechanism (Chappa et al., 2006) and CGRP can be drained off into the venous blood plasma after it is released from trigeminal afferents (Messlinger, 2018). It was demonstrated that VIP crosses the BBB unidirectionally from blood to brain via transmembrane diffusion (Dogrukol-Ak et al., 2003) but there is yet no evidence for a reverse situation. However, these neuropeptides may also be drained off into the venous blood plasma as the result of increased permeability of BBB due to vasopermeability-enhancing neuropeptide SP or neuroinflammation (Levy et al., 2018; Ramachandran, 2018). However, cervical sympathectomy treatment attenuated SAH-induced increases in plasma levels of CGRP, SP and VIP suggesting that cervical sympathectomy may ameliorate neuroinflammation in the nervous system after SAH. In an experimental study, it was reported that cervical sympathectomy decreased neurogenic vasodilation in cranial dura mater in rats (Wei et al., 2011). Our findings are in concordance with this study. On the other hand, a previous study reported that SAH decreased plasma CGRP concentrations, whereas the blockade of sympathetic ganglion following SAH increased it in the experimental SAH model in rats (Hu et al., 2014). The plasma CGRP findings in our study were completely contrary to that ones in the study, but one 6
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therefore they may be diluted in total brain tissue. Moreover, main source of CGRP, SP and VIP are mostly cell bodies of neurons in trigeminal ganglion. Another possible explanation may be that CGRP, SP and VIP in the brain tissue may be expeditiously broken down or removed. Moreover, our findings revealed that the sympathectomy improved the neurological symptoms impaired by SAH. Someone can ask how did the sympathectomy improve the neurological symptoms without changing CGRP, SP and VIP concentrations in the brain tissue. In addition to above mentioned possible reasons may be valid for this, the sympathectomy may have protective effect against brain injury by attenuating oxidative and nitrative stress products and pro-inflammatory cytokines in the brain tissue as a result of prevention of cerebral vasospasm. On the other hand, the main finding of our study is that cervical sympathectomy following SAH reduced the SAH-induced c-fos expression in TNC, and levels of neuropeptides in the plasma, trigeminal ganglion and brainstem. This finding suggests that cervical sympathectomy treatment may be a promising therapeutic approach in the treatment of neuroinflammation in surviving patients with SAH. The current treatments for SAH contain hemodynamic augmentation and medically or surgically performed intra-arterial vasodilation (van Gijn and Rinkel, 2001). But, these approaches provide relatively less effect on the delayed neurological deteriorations. We can speculate that after such treatments, basal and evoked sympathetic discharges will still continue unless cervical sympathetic ganglion are blocked. This is a massive risk factor for surviving patients. Because SAH-induced local tissue mediators plus a small sympathetic stimulation could lead to nearly closure of cerebral and meningeal arteries. It was reported in clinical trials that blockade of different sympathetic ganglions can be carried out for the treatment of various conditions such as complex regional pain syndrome type I (reflex sympathetic dystrophy), causalgia, cluster or vascular headache, phantom limb pain, post-stroke pain, refractory angina pectoris, hyperhidrosis, Raynaud's syndrome, idiopathic livedo reticularis etc. (Mailis and Furlan, 2010; Bandyk et al., 2002; Ebrahim, 2011; Lowell et al., 1993). Sympathetic blockade can be performed in two different ways including chemical and surgical sympathectomy. While chemical sympathectomy has temporary therapeutic effects due to over time degradation of injected chemical agents, the effects of surgical sympathectomy is permanent and safer. Blockade of different sympathetic ganglions (like stellate ganglion) exhibits therapeutic effects by improving the blood supply of the region, and reducing plasma concentration of adrenal hormones. Above mentioned reports from the clinical trials suggested that surgical sympathectomy has therapeutic effects for the treatment of those painful conditions, and it exhibits unimportant side effects. Thus, cervical sympathectomy treatment may prevent early brain injury due to enhanced neurogenic inflammatory neuropeptides such as CGRP, SP and VIP, and consequently deficits in patients with SAH who were arrived to the hospital before occurrence of early brain injury. Here, it can be inquired whether sympathetic denervation of cerebral arteries as a result of the sympathectomy will adversely affect cerebral circulation. However, since the vasomotion of cerebral arteries/arterioles after sympathetic blockage can be mediated by mediators including local tissue factors, there may probably not be any disruption in the cerebral circulation in the long term. Furthermore, the sympathectomy may improve the life quality of patients by suppressing neurogenic inflammatory reactions in patients with SAH, who are late to hospital or survived after SAH. Therefore, surgical cervical sympathectomy treatment may be a good physiological treatment approach as an alternative to drug therapy after SAH.
However, cervical sympathectomy treatment reduced SAH-induced the increases in CGRP, SP and VIP levels in trigeminal ganglion and CGRP and SP levels in the brainstem. These findings suggest that the sympathectomy treatment shortly after SAH may prevent neurogenic inflammation at origin site of the inflammation. The superior cervical ganglion supplies sympathetic innervation to cerebral and meningeal arteries (Lv et al., 2014). There is a basal sympathetic nerve discharge to these vessels by cervical ganglion to maintain basal tonus of the vessels even if there is not any stimulus. Together basal tonus of the vessels due to basal sympathetic discharge plus vasospasm following SAH can lead to delayed brain deterioration, delayed cerebral ischemia, delayed neurological deficits and chronic head pain after SAH. Moreover a sympathetic activation over basal level after SAH could further promote cerebral vasospasm and correspondingly more neurological deteriorations could occur. In an experimental study, it was shown that vasoconstriction in middle and basal cerebral arteries occured following induction of experimental SAH in rats, however, this vasospasm was reduced by cervical sympathectomy application (Hu et al., 2014). However, (neuronal) cell body response approach including the changes in expression of certain neuropeptides in peripheral neurons after the axotomy may explain possible mechanism underlying the effect of cervical sympathectomy treatment after SAH in the present study. It is well established that chemical or surgical sympathectomy (axotomy) results in changes in gene expression of certain neuropeptides in peripheral neurons in vivo (Zigmond, 2012). These changes contain increases in expression of certain neuropeptides and decreases in some other neuropeptides (Zigmond, 2012). However, the changes for a neuropeptide can include either increase or decrease in different neural tissues. For instance, while SP increases in sympathetic and motor neurons following axotomy it reduces in sensory ganglia (HyattSachs et al., 1996). In addition, Zigmond’s group reported that chemical sympathectomy and postganglionic axotomy lead to increases in VIP mRNA levels in the rat superior cervical ganglia (Hyatt-Sachs et al., 1996; Mohney et al., 1994). Moreover they demonstrated that axotomy of sensory neurons in lumbar dorsal root ganglia reduced protein and mRNA levels of neuropeptides CGRP and SP in this ganglia (Shadiack et al., 2001). On the other hand, it was previously shown that axonal sprouts contain CGRP and SP in the dorsal root ganglia after sciatic nerve transection in rats (McLachlan and Hu, 1998). In the present study, we measured the concentrations of the neuropeptides in plasma and different tissues including trigeminal ganglia, brainstem and brain. Thus, in the present study, it can be queried whether cervical sympathectomy treatment after SAH may have decreased levels of neurogenic inflammatory neuropeptides CGRP, SP and VIP through the cell body response. Since we surgically excised superior cervical ganglia that contain the cell bodies of neurons contrary to the axons, it seems unlikely that cervical sympathectomy act as directly via this mechanism. However it may acts as indirectly via an unknown mechanism on the levels of these neuropeptides. Another question may be whether the changes occured in SAH plus sympathectomy can also happen in only sympathectomy if sympathectomy alone was carried out. It would be favorable if it was carried out also sympathectomy alone but this does not adversely affect our current results. Because sympathectomy treatment in current study didn’t include the axotomy and we used both naive and saline controls. However, in here we first showed that cervical sympathectomy alleviates SAH-induced neurogenic inflammatory neuropeptides CGRP, SP and VIP in the plasma, trigeminal ganglion and brainstem. Therefore, we believe that our findings can be able to pioneer further comprehensive studies on the effects of sympathectomy treatment following SAH. In addition, in the present study, neither SAH nor the sympathectomy had any effect on the CGRP, SP and VIP levels in total brain tissue. The most likely reason for this may be that the concentrations of these neuropeptides were measured from total brain tissue and
3.1. Limitations of the study In addition to immunohistochemical staining method that we used in the present study to determine c-fos positive-cells in TNC in the brain stem, it would be favorable if it was carried out also quantitative 7
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tilted caudally at a 30° angle over 20 s. The rats in the saline group were injected with equal volume of sterile saline in the same way. The burr hole was sealed with bone wax. The rats were injected (i.p.) with 1 ml of saline to prevent dehydration (i.p.) and then they were placed in the cages with a 30° head-down position, therefore, blood was provided to distribute around the basal arteries for 30 min. Two rats in the SAH + Symp group that died during surgical operations were excluded from the experiments.
Western blot or ELISA technique. Moreover if it was carried out also sympathectomy group alone, it would have further enriched current findings. 4. Materials and methods 4.1. Animals The animals used in the study were treated according to the Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011, The National Academies Press). Male Wistar rats weighing 200–250 g were used in the experiments. Rats were purchased from Abant Izzet Baysal University, the experimental animals implementation and research centre (Turkey). The rats were fed ad libitum with rodent food and water, and were housed in their individual cages at a room temperature (22 ± 2 °C) and a 12 h light/dark cycle. All experimental procedures were approved by Abant Izzet Baysal University, Local Ethics Committee for Animal Experiments (license number 2016/28).
5.2. Establishment of the TCSG model
Male Wistar rats (10 weeks old) were randomly divided into 4 groups: the control group (n = 7), no procedure was performed; Saline group (n = 14) was injected with 300 μl of normal saline into the prechiasmatic cistern; SAH + Sham group (n = 14) was exposed to induction of subarachnoid hemorrhage; SAH + Symp group (n = 14) was exposed to left transection of the cervical sympathetic ganglion following SAH induction. Moreover, for c-fos immunohistochemical staining, seven rats were used for each group. Experimental groups and surgical interventions are shown in Table 1.
Transection of the cervical sympathetic ganglion (TCSG) was carried out in rats in SAH + Symp group. One hour after induction of SAH, the left superior cervical sympathetic ganglion was removed as previously described by Kilinc et al. (Kilinc et al, 2015). Under still ketamine and xylazine anaesthesia (above mentioned) and under a dissecting microscope, a 1-cm incision was made aseptically in the ventral area of the neck, left sternocleidomastoid muscle was exposed and deflected, then the bifurcation of the left carotid artery were seen. Because of the left superior cervical ganglion is placed under bifurcation of the left carotid artery, the left carotid artery was displaced laterally to expose the superior cervical ganglion. Then, the superior cervical ganglion with pre- and post-ganglionic fibers was cut and removed via a fine-scissors and forceps, respectively. Adequate care was endeavoured not to damage the adjacent vagal and hypoglossal nerves. The incision was sutured under aseptic conditions. Success of transection of the cervical sympathetic ganglion was confirmed by ipsilateral ptosis (droopy eyelid). Left cervical sympathetic ganglions in the Saline and SAH+Symp groups were exposed but not transected. After surgery interventions, rats in all groups were kept alive for 14 days under appropriate conditions.
5. Materials
5.3. Neurological evaluation
Paraformaldehyde and phosphate-buffered saline were purchased from Sigma-Aldrich, (Schnelldorf, Germany), c-fos kit was purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA), CGRP, SP and VIP ELISA kits were purchased from ELABscience (Wuhan, P.R. China).
Neurological functions of the rats were assessed by a blinded investigator at 2th, 8th and 14th days following induction of SAH as previous described (Ersahin et al., 2009). In summary, a 20-point neurological score was used to evaluate motor and behavioral deficits. The motor and behavioral performance were graded in a score range of 0–20. Higher scores indicate severe neurological deficits.
4.2. Experimental groups
5.1. Induction of experimental SAH The experimental SAH model was constructed according to the procedure described in a previous study, which included the injection of autologous blood into the prechiasmatic cistern (Prunell et al., 2002). Briefly, after the rats were anesthetized with the intraperitoneal (ip) injection of ketamine (90 mg/kg, Ketalar; Pfizer, New York, NY) and xylazine (5 mg/kg; Rompun; Bayer, Leverkusen, Germany), they were placed prone in a stereotactic apparatus (Stoelting Europe, Dublin, Ireland). In aseptic conditions, a small incision was made in the midline to the scalp and a 1 mm hole was drilled in the skull 7.5 mm anterior to the bregma in the midline without damaging the dura mater. Then, 300 μl of autologous blood from the femoral artery was injected gently into the prechiasmatic cistern through a 26 G spinal needle which was
5.4. Blood collection, harvesting and homogenization of the tissues 14 days after surgical interventions, under anesthesia with ketamine (90 mg/kg, i.p.) thorax of rats was opened by a scissor, and blood samples were taken from the right ventricle by cardiac puncture. Rats, excluding used for c-fos immun-staining, were perfused intracardially with only 100 ml PBS (phosphate-buffered saline, pH: 7.4) to remove blood from structures of the nervous system. The head was separated from the body at the atlanto-occipital joint, the brain was removed. Brainstem including trigeminal nucleus caudalis area was dissected within the following limits: coronal sections from −9 to −15 mm posterior to bregma, 4 mm lateral to bregma (Paxinos’ the rat brain
Table 1 Experimental groups and surgical interventions. Group
Surgical interventions
Final number of animals
Control Saline
not touched (to determine naive concentrations of c-fos, CGRP, SP and VIP) 300 μl of normal saline was injected into the prechiasmatic cistern. One hour after saline injection, left cervical sympathetic ganglion were exposed but not transected 300 μl of autologous blood was injected into the prechiasmatic cistern. One hour after blood injection, left cervical sympathetic ganglion were exposed but not transected 300 μl of autologous blood was injected into the prechiasmatic cistern. One hour after blood injection, left superior cervical sympathetic ganglion was removed
7 14
SAH + Sham SAH + Symp
SAH: subarachnoid hemorrhage; Symp: sympathectomy. 8
14 14
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mean ± standard error of the mean. Statistical analysis was performed using SPSS for Windows (version 17.0, SPSS Inc., Chicago, IL, USA). The statistical differences between the different groups were analyzed by one-way analysis of variance (ANOVA) and followed by LSD test for multiple comparisons. A value of p < 0.05 was considered statistically significant.
atlas), with a depth of 2–3 mm. Trigeminal ganglia were dissected by cutting the nerve divisions approximately 2 mm proximal and distal to the branching point of the mandibular nerve. The dura was intently removed from the ganglia under stereo-microscope. The brain, brainstem and trigeminal ganglia samples were put into the tubes, and the tubes were immediately placed in liquid nitrogen. Blood samples were centrifuged at 3000 × g for 15 min at 4 °C. Homogenization of brain tissue, brainstem and trigeminal ganglia samples was performed using a light-duty Ultra-Turrax homogenizer (ISOLAB, Wertheim, Germany) as previously described by Kilinc et al. (2018). The supernatants were stored at −80 °C until assayed for CGRP, SP and VIP immunoreactivities by ELISA. On the other hand, rats to be used for immunohistochemical staining for c-fos expression were perfused intracardially with 100 ml PBS (phosphate-buffered saline, pH: 7.4) and followed by 200 ml 4% paraformaldehyde (PAF). Brainstem samples were incubated overnight in 4% PAF.
Acknowledgements We declare that there are not any relationships that present a potential conflict of interest. We thank Shawnda Camsari who carried out the proofreading of this article in terms of English grammar. Funding This study was supported by Abant Izzet Baysal University Scientific Research Fund (Grant number: 2016.08.09.1096).
5.5. Determination of CGRP, SP and VIP concentrations in plasma and the tissues
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Research report
Cervical sympathectomy modulates the neurogenic inflammatory neuropeptides following experimental subarachnoid hemorrhage in rats☆
T
⁎
Yasar Dagistana, Erkan Kilincb, , Cemre Nur Balcic a
Abant Izzet Baysal University, Medical Faculty, Department of Neurosurgery, Bolu, Turkey Abant Izzet Baysal University, Medical Faculty, Department of Physiology, Bolu, Turkey c Abant Izzet Baysal University, Institute of Health Sciences, Department of Histology and Embryology, Bolu, Turkey b
H I GH L IG H T S
hemorrhage induces c-fos expression in the trigeminal nucleus caudalis. • Subarachnoid attenuates subarachnoid hemorrhage-induced c-fos expression. • Sympathectomy hemorrhage induces release of neurogenic inflammatory neuropeptides. • Subarachnoid • Sympathectomy modulates induced release of neurogenic inflammatory neuropeptides.
A R T I C LE I N FO
A B S T R A C T
Keywords: Subarachnoid hemorrhage Cervical sympathectomy Neuropeptides Neurogenic inflammation Trigeminovascular system
Background: Neuroinflammation is implicated in cerebral vasospasm and brain injuries after subarachnoid hemorrhage (SAH). In addition to classical neuroinflammation with increased inflammatory cytokines, a sterile neurogenic inflammation characterized by release of potent vasoactive neuropeptides may be responsible for brain injuries after SAH. Sympathetic discharges from superior cervical ganglion contribute to vasoconstriction of cerebral arteries Thus, we investigated the effects of surgical cervical sympathectomy on the neurogenic inflammatory neuropeptides shortly after SAH induction in a model of SAH in rats. Methods: Male Wistar rats were divided into 4 groups: control; was not touched, saline group; 300 μl of saline was injected into prechiasmatic cistern, SAH+Sham group; 300 μl of autologous blood was injected to induce subarachnoid hemorrhage into prechiasmatic cistern; SAH+Symp group; the left cervical sympathetic branch was surgically removed after the induction of SAH. Levels of neuropeptides CGRP, SP and VIP which are responsible for neurogenic inflammation, in plasma, trigeminal ganglion, brainstem and brain tissue were measured by ELISA. In addition, c-fos expression as a marker of neuronal activation in the trigeminal nucleus caudalis (TNC) was determined by immunohistochemical staining. Results: SAH significantly increased c-fos expression in the TNC, as well as CGRP, SP and VIP concentrations in plasma and trigeminal ganglion neurons, and also CGRP and SP concentrations in the brainstem. Cervical sympathectomy application significantly reduced the increases in these parameters induced by SAH. Conclusions: Our findings suggest that cervical sympathectomy treatment may prevent early brain injury by modulating SAH-induced neurogenic inflammatory neuropeptides such as CGRP, SP and VIP, and improve the quality of life in survivors following SAH.
1. Introduction Subarachnoid hemorrhage (SAH) is a life-threatening neurological problem with 50% of pre-hospital deaths (Campos-Pires et al., 2016). The most common cause of SAH, except for trauma is the rupture of an
aneurysm in the brain (Goksu et al., 2016). It has been reported that approximately 75% of patients with aneurysmal SAH die and the most survivors are severely disabled (Hop et al., 1997). Sudden and severe headache is the most common warning symptom of SAH and it chronically persists in the survivors after SAH (Ferro and Melo, 1998;
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Presentation at a conference: This study has been presented as oral presentation at 15th Pain Congress with International Participation on November 15 – 18, 2018, in Antalya, Turkey. ⁎ Corresponding author. E-mail address: [email protected] (E. Kilinc). https://doi.org/10.1016/j.brainres.2019.146366 Received 9 March 2019; Received in revised form 6 August 2019; Accepted 7 August 2019 Available online 08 August 2019 0006-8993/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Neurological scores after subarachnoid hemorrhage. (A) Neurological score of each group at 2th, 8th and 14th days post-SAH, (B) mean neurological score of groups at 2th, 8th and 14th days postSAH. **P < 0.01 (SAH + Sham vs Saline, and ##P < 0.01 (SAH + Symp vs SAH + Sham). SAH: subarachnoid hemorrhage; Symp: sympathectomy.
was reported that vasoconstriction in middle and basal cerebral arteries occured following induction of experimental SAH in rats, however, this vasospasm was reduced by cervical sympathectomy application (Hu et al., 2014). Therefore, in the present study, we aimed to investigate the effects of postoperative superior cervical sympathectomy on the cfos expression, a marker of neuronal activation, in the trigeminal nucleus caudalis and levels of neuropeptides (CGRP, SP and VIP) leading to neurogenic inflammation in the plasma and strategical structures (trigeminal ganglion, brainstem and brain tissue) related to their expression and release in a model of SAH in rats.
Chen and Ayata, 2016). It is well established that non-infectious neuroinflammation plays a key role in the pathophysiology of SAH (Zheng and Wong, 2017). Current data report that this neuroinflammation contributes to early brain injury, cerebral vasospasm and consequently delayed neurological deterioration and the disabilities after SAH (Zheng and Wong, 2017; Lucke-Wold et al., 2016; de Oliveira Manoel and Macdonald, 2018). In addition to classical neuroinflammation with increased inflammatory cytokines such as IL-1β, IL-6, TNF-α etc, a sterile neurogenic inflammation characterized by the release of potent vasoactive neuropeptides can occur following SAH. Vasoactive neuropeptides including calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP) are considered to be key mediators in neurogenic inflammation (Lewis et al., 2013; Corrigan et al., 2016; Messlinger et al., 2011; Schytz et al., 2010). It was suggested that neurogenic inflammatory response can exacerbate classical neuroinflammation via a positive feedback loop with classical inflammatory mediators (Corrigan et al., 2016). However, inflammation and oxidative stress mediators, and protons following SAH can induce both expression of vasoactive neuropeptides in trigeminal ganglion neurons and their release from peripheral and central terminals of trigeminal nerve fibers in the cranial dura mater and brainstem, respectively (Berczi et al., 2010; Ichikawa and Sugimoto, 2002; Raddant and Russo, 2014). Moreover, increased intracranial pressure due to SAH may also lead to release of these vasoactive neuropeptides by activating mechanoreceptors in trigeminal afferent nerve terminals innervating meninges due to these neuropeptides-expressing neurons are sensitive to noxious mechanical stimuli (Iyengar et al., 2017). Upon released, CGRP and VIP cause vasodilation of cerebral blood vessels, however, SP leads to extravasation of plasma proteins (Levy et al., 2018; Ramachandran, 2018). As a result of all of these events, a sterile neurogenic inflammation in the brain and meninges occurs. Interestingly, CGRP and SP released from trigeminal sensory nerve fibers innervating meningeal blood vessels further promote neurogenic inflammation by activating mast cells, which are resident immune cells in the meninges (Ottosson and Edvinsson, 1997; Ramachandran, 2018; Strassman et al., 1996; Theoharides et al., 2005). In addition, CGRP and SP also induce the initiation of headache due to neurogenic inflammation by sensitizing sensory nerve terminals and they facilitate transmission of pain signals to second-order neurons in trigeminal nucleus caudalis (TNC) in the brainstem (Strassman et al., 1996), where it harbours central terminals of trigeminal afferent nerve fibers. The superior cervical ganglion is a part of the sympathetic nervous system and supplies sympathetic innervation to the head and neck. It
2. Results 2.1. TCSG treatment ameliorated neurological function in rats with SAH It was not observed animal deaths in the sham group, but the mortality rate of rats in SAH group was bigger at 48 hr (6/20; 30%) than that in SAH + Symp group (4/18; 22.2%). The neurological scores at 2th days post-SAH for control group was 0 ± 0, for Saline group was 0.42 ± 0.2, for SAH + Sham group was 10.8 ± 0.59, and for SAH + Symp group was 5.8 ± 0.45. The neurological scores at 8th days post-SAH for control group was 0 ± 0, for Saline group was 0.28 ± 0.18, for SAH + Sham group was 11.5 ± 0.36, and for SAH + Symp group was 6.4 ± 0.52. The neurological scores at 14th days post-SAH for control group was 0 ± 0, for Saline group was 0.14 ± 0.1, for SAH + Sham group was 10.5 ± 0.57, and for SAH + Symp group was 5.7 ± 0.52. Mean values of the neurological scores at these 3 different set point for control group was 0 ± 0, for Saline group was 0.28 ± 0.15, for SAH + Sham group was 11 ± 0.34, and for SAH + Symp group was 6 ± 0.39. The neurological scores at 2th, 8th, and 14th days post-SAH in SAH + Sham group rats were higher compared to the Saline group, respectively (P = 0.004, P = 0.002 and P = 0.005, Fig. 1A). Following TCSG, these rats showed a significant reducing in motor and behavioral deficits compared to the SAH + Sham groups, respectively (SAH + Symp vs SAH + Sham, P = 0.009, P = 0.004 and P = 0.008, Fig. 1A). Likewise, in terms of mean values of the neurological scores at these 3 different set point, rats in SAH + Sham group showed higher the neurological scores than that in the Saline group (SAH + Sham vs Saline, P = 0.0021, Fig. 1B), and following TCSG, these rats exhibited a significant reducing in motor and behavioral deficits (SAH + Symp vs SAH + Sham, P = 0.0041, Fig. 1B). 2
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Fig. 2. Effects of subarachnoid hemorrhage induction and sympathectomy treatment on the c-fos expression in trigeminal nucleus caudalis in the brainstem. (A) c-fos positive neurons in trigeminal nucleus caudalis in control group, (B) in saline group, (C) in SAH + Sham group and (D) in SAH + Symp group, (E) while SAH increased the number of c-fos positive neurons, the sympathectomy treatment reduced these increases in trigeminal nucleus caudalis in the brainstem (the number of c-fos positive neurons is given as per section in the graph). Arrowheads demonstrate c-fos positive neurons in trigeminal nucleus caudalis. Please note, increased the number of c-fos positive neurons in the SAH + Sham group compared to saline, and decrease in SAH + Symp group compared to SAH + Sham group. ***P < 0.001 (SAH + Sham vs Saline), and #P < 0.05 (SAH + Symp vs SAH + Sham). SAH: subarachnoid hemorrhage; Symp: sympathectomy.
290.8 ± 19 pg/ml to 212.3 ± 18.4 (SAH + Symp vs SAH + Sham, P = 0.003, Fig. 4A) and VIP concentrations from 29.1 ± 2.9 pg/ml to 21.3 ± 1.2 (SAH + Symp vs SAH + Sham, P = 0.028, Fig. 5A) in the plasma respectively, compared to SAH.
2.2. TCSG reversed up-regulation of c-fos protein expression in TNC The increase in c-fos expression in TNC is a key marker of neuronal activation that indicates also the activation of trigeminovascular system (Bullitt, 1990). In addition, neurogenic inflammation plays a key role in activation and sensitization of the trigeminovascular system (Levy, 2012). Thus we explored c-fos expression in TNC in experimental SAH model in current study. In the present study, the number of c-fos positive neurons in the TNC were 13 ± 1.0 in per section in control group (Fig. 2A and E). However, SAH increased the number of c-fos positive neurons in the TNC from 15 ± 1.0 to 43 ± 1.9 in per section compared to the Saline group (SAH + Sham vs Saline, P = 0.001, Fig. 2B, C and E), on the other hand, TCSG treatment in the SAH + Symp group reduced SAH-induced the increase in the number of c-fos positive neurons from 43 ± 1.9 to 34 ± 1.7 in per section compared to SAH + Sham group (P = 0.011, Fig. 2C, D and E).
2.5. TCSG reversed up-regulation of CGRP, SP and VIP concentrations in trigeminal ganglion SAH increased CGRP concentrations from 55.8 ± 3.3 pg/mg/ml to 73.9 ± 4.2 pg/mg/ml (SAH + Sham vs Saline, P = 0.004, Fig. 3B) and SP concentrations from 434.3 ± 60.4 pg/mg/ml to 786.6 ± 34.9 pg/ mg/ml (SAH + Sham vs Saline, P = 0.001, Fig. 4B) and VIP concentrations from 85.8 ± 4.9 pg/mg/ml to 145.8 ± 22.2 pg/mg/ml (SAH + Sham vs Saline, SAH + Sham vs Saline, P = 0.011, 5B) in trigeminal ganglion respectively, compared to Saline group. On the other hand, TCSG treatment decreased CGRP concentrations from 73.9 ± 4.2 pg/mg/ml to 60.3 ± 2.3 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.037, 3B) and SP concentrations from 786.6 ± 34.9 pg/mg/ml to 523.0 ± 51.8 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.005, 4B) and VIP concentrations from 145.8 ± 22.2 pg/mg/ml to 96.9 ± 9.2 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.047, 5B) in trigeminal ganglion respectively, compared to SAH + Sham group.
2.3. Effect of TCSG on vasoactive neuropeptides in SAH rats CGRP, SP and VIP are potent vasoactive neuropeptides that play key roles in the neurogenic inflammation (Lewis et al., 2013; Corrigan et al., 2016; Messlinger et al., 2011; Schytz et al., 2010). Therefore, in present study, we measured the concentrations of these vasoactive neuropeptides in plasma and in the structures related to their expression and release including trigeminal ganglion, brainstem and brain tissue.
2.6. TCSG reversed up-regulation of CGRP and SP concentrations in brainstem
2.4. TCSG reversed up-regulation of CGRP, SP and VIP concentrations in plasma
While SAH increased CGRP concentrations from 25.1 ± 1.7 pg/ mg/ml to 41.7 ± 4.1 pg/mg/ml (SAH + Sham vs Saline, P = 0.005, Fig. 3C) and SP concentrations from 321.1 ± 38.9 pg/mg/ml to 593.1 ± 73.4 pg/mg/ml (SAH + Sham vs Saline, P = 0.003, Fig. 4C), it did not change VIP concentrations (from 49.4 ± 3.4 pg/mg/ml to 56.7 ± 4.8 pg/mg/ml, SAH + Sham vs Saline, P = 0.65, Fig. 5C) in brainstem, respectively compared to saline group. On the other hand, TCSG treatment decreased CGRP concentrations from 41.7 ± 4.1 pg/ mg/ml to 29.8 ± 3.3 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.04, Fig. 3C) and SP concentrations from 593.1 ± 73.4 pg/mg/ml to 369.8 ± 36.7 pg/mg/ml (SAH + Symp vs SAH + Sham, P = 0.016,
SAH increased CGRP concentrations from 24.6 ± 1.0 pg/ml to 37.1 ± 1.3 pg/ml (SAH + Sham vs Saline, P = 0.001, Fig. 3A) and SP concentrations from 151.9 ± 7.2 pg/ml to 290.8 ± 19 pg/ml (SAH + Sham vs Saline, P = 0.001, Fig. 4A) and VIP concentrations from 17.2 ± 1.3 pg/ml to 29.1 ± 2.9 pg/ml (SAH + Sham vs Saline, P = 0.001, Fig. 5A) in the plasma respectively, compared to Saline group. On the other hand, TCSG treatment decreased CGRP concentrations from 37.1 ± 1.3 pg/ml to 30.4 ± 0.6 pg/ml (SAH + Symp vs SAH + Sham, P = 0.001, Fig. 3A) and SP concentrations from 3
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Fig. 3. Effects of subarachnoid hemorrhage induction and sympathectomy treatment on CGRP concentrations in the plasma, trigeminal ganglion, brainstem and brain tissue. **P < 0.01, ***P < 0.001, #P < 0.05, ###P < 0.001. *: SAH + Sham vs Saline, #: SAH + Symp vs SAH + Sham.
brain injury after SAH (de Oliveira Manoel and Macdonald, 2018; Zheng and Wong, 2017; Lucke-Wold et al., 2016). 30% of patients, survivors following SAH, are exposed to delayed brain deterioration (Zheng and Wong, 2017). Delayed brain deterioration commonly shows up 3–14 days later and it is characterized by delayed cerebral ischemia and delayed neurological deficits (Chen et al., 2015). Cerebral vasospasm that appears in patients with SAH who survive following aneurysmal or non-aneurysmal SAH leads to the ischemic brain injury, and an increase in the level of pro-inflammatory cytokines and reactive oxygen species in the brain (Denes et al., 2010; Fujii et al., 2013). The resulting neroinflammation can cause the release of vasoactive and pro-nociceptive neuropeptides promoting the neurogenic inflammation such as CGRP, SP and VIP through axon reflex by stimulating receptors in trigeminal sensory nerve terminals, which are innervating the cranial dura mater (Levy et al., 2018; Lukács et al., 2015; Ramachandran, 2018). While CGRP and VIP cause the vasodilatation of meningeal blood vessels, SP gives rise to plasma protein extravasation by increasing the permeability of meningeal blood vessels (Levy et al., 2018; Ramachandran, 2018). Moreover, each of neuropeptides CGRP, SP and VIP also leads to the release of numerous inflammatory mediators from the mast cells in the cranial dura mater by degranulating them (Levy et al., 2018; Ottosson and Edvinsson, 1997; Theoharides et al., 2012). Mast cell activation would in turn further enhance dural neurogenic inflammation (Theoharides et al., 2012) As a domino,
Fig. 4C) in brainstem, respectively compared to SAH + Sham group. 2.7. SAH or TCSG did not change CGRP, SP and VIP concentrations in brain tissue Interestingly, neither induction of SAH nor TCSG treatment changed CGRP, SP and VIP concentrations in brain tissue. CGRP concentrations were 38.6 ± 3.0 pg/mg/ml in Saline group, 41.4 ± 4.5 pg/mg/ml SAH + Sham group, 40.0 ± 3.9 pg/mg/ml in SAH + Symp group; P = 0.93 for Saline group vs SAH + Sham (Fig. 3D), P = 0.99 for SAH + Sham versus SAH + Symp (Fig. 3D). SP concentrations were 270.3 ± 15.7 pg/mg/ml in Saline group, 287.6 ± 38.5 pg/mg/ml SAH + Sham group, 272.0 ± 20.9 pg/mg/ml in SAH + Symp group, P = 0.95 for Saline group vs SAH + Sham (Fig. 4D), P = 0.96 for SAH + Sham versus SAH + Symp (Fig. 4D). VIP concentrations were 116.0 ± 8.0 pg/mg/ml in Saline group, 127.4 ± 12.0 pg/mg/ml in SAH + Sham group, 118.9 ± 6.6 pg/mg/ml in SAH + Symp group, P = 0.83 for Saline group vs SAH + Sham group (Fig. 5D), P = 0.92 for SAH + Sham group vs SAH + Symp group(Fig. 5D). 3. Discussion Neuroinflammation following SAH contributes to early brain injury and then neuroinflammation processes participate in the secondary 4
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Fig. 4. Effects of subarachnoid hemorrhage induction and sympathectomy treatment on SP concentrations in the plasma, trigeminal ganglion, brainstem and brain tissue. **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01. *: SAH + Sham vs Saline, #: SAH + Symp vs SAH + Sham.
et al., 1998; Ostergaard, 1991; Schwedt and Matharu, 2006). Therefore, in our study, the sympathectomy treatment decreased SAH induced cfos expression suggesting that the sympathectomy treatment may alleviate the overactivation of trigeminovascular system and prevent chronic headache after SAH. The potent vasoactive neuropeptides CGRP, SP and VIP are closely associated with the generation of dural neurogenic inflammation (Schytz et al., 2010; Ramachandran, 2018). CGRP, SP and VIP are released upon activation of the trigeminal ganglion or fibers, and this release of the neuropeptides initiate a sterile neurogenic inflammation in the meninges (Messlinger et al., 1993; Moskowitz, 1993). Moreover this neurogenic inflammation also leads to the activation of mast cells in the cranial dura mater. Then dural mast cells further promote neurogenic inflammation by release of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, IFN-β (Theoharides et al., 2012; Conti et al., 2017; Caraffa et al., 2018) from their cytoplasmic granules. Therefore, it can be stated that CGRP, SP and VIP are chief triggers for dural neurogenic inflammation since they propagate neurogenic inflammation by activating immune mast cells in the meninges. Therefore in current study, we target the neurogenic inflammatory neuropeptides including CGRP, SP and VIP apart from the inflammatory cytokines in the classical inflammation. In the present study, we found that SAH increased plasma CGRP, SP and VIP concentrations. These findings are important due to the fact that increased plasma levels of these neuropeptides may be an indirect
neuroinflammation and cerebral vasospasm following SAH can evoke delayed brain deterioration, delayed cerebral ischemia, delayed neurological deficits and chronicity of headache after SAH. Therefore, in particular prevention of cerebral vasospasm after SAH may prevent neurogenic inflammation and by extension the other related impairments. In the direction of this hypothesis, in the present experimental study, we performed cervical sympathectomy following induction of SAH in the experimental model of SAH, and found that the sympathectomy reduced the increases in markers of neuronal activation and dural neurogenic inflammation including c-fos, CGRP, SP and VIP evoked by SAH. C-fos expression as a marker of neuronal activation in the brainstem is widely used to investigate neuronal activation based on disturbance of meninges in experimental studies (Kilinc et al., 2018; Zhang et al., 2017). In the present study, we found that c-fos expression was increased in TNC in SAH group. This finding is extremely important because it is well known that the trigeminovascular system is activated during head pain stemming from meningeal irritation by dural inflammation (Kilinc et al., 2017; Levy et al., 2018; Ramachandran, 2018). Thus, in our study, the increase in c-fos expression in SAH group indicates that SAH leads to the activation of the trigeminovascular system. The activation of the trigeminovascular system may be a sign of generation and transmission of head pain after SAH. Moreover, this finding is consistent with previous studies reporting that the rupture of a saccular intracranial aneurysm leads to thunderclap headache (Linn 5
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Fig. 5. Effects of subarachnoid hemorrhage induction and sympathectomy treatment on VIP concentrations in the plasma, trigeminal ganglion, brainstem and brain tissue. *P < 0.05, **P < 0.01, #P < 0.05. *: SAH + Sham vs Saline, #: SAH + Symp vs SAH + Sham.
possible reason for this may be that our investigation is a longer-term study covering 14 days. Because the symptoms of vasospasm based on SAH develop slowly throughout the time. In addition, in the present study, SAH increased the levels of CGRP, SP and VIP also in trigeminal ganglion neurons and the levels of CGRP and SP in the brainstem which are key structures indicating a painful inflammation in the brain and meninges. These findings are also in accord with the increase in the c-fos expression in TNC in the brainstem in the current study because of up-regulation in c-fos expression indicates transmission of pain signals from the head with neuroinflammation due to activation of the trigeminovascular system (Levy, 2012; Bullitt, 1990). Our these findings are important due to the fact that neuropeptides CGRP, SP and VIP released from meningeal nerve terminals are mostly expressed in the soma of trigeminal ganglion neurons (Goto et al., 2017; Lazarov, 2002). Moreover these findings suggest that SAH may evoke neurogenic inflammation through also activation of trigeminal ganglion neurons. In addition, SAH increased CGRP and SP levels without changing VIP levels in the brainstem. In this study, the increase in CGRP and SP levels in the brainstem indicates most probably a painful neuroinflammation of the brain and meninges. Moreover, SAH may lead to neurogenic inflammation not only in the meninges and brain but also in the brainstem via an increase CGRP and SP levels in the brainstem. On the other hand, SAH did not change VIP levels in the brainstem. This may be due to VIP expression in TNC is relatively less compared to those in trigeminal ganglion neurons.
reflection of neuroinflammation in the nervous system. Because, it was previously demonstrated that trigeminal ganglion, which is the major source of these neuropeptides, is outside of the blood–brain barrier (BBB) (Eftekhari et al., 2015). Moreover it was reported that SP can cross BBB through a carrier-mediated mechanism (Chappa et al., 2006) and CGRP can be drained off into the venous blood plasma after it is released from trigeminal afferents (Messlinger, 2018). It was demonstrated that VIP crosses the BBB unidirectionally from blood to brain via transmembrane diffusion (Dogrukol-Ak et al., 2003) but there is yet no evidence for a reverse situation. However, these neuropeptides may also be drained off into the venous blood plasma as the result of increased permeability of BBB due to vasopermeability-enhancing neuropeptide SP or neuroinflammation (Levy et al., 2018; Ramachandran, 2018). However, cervical sympathectomy treatment attenuated SAH-induced increases in plasma levels of CGRP, SP and VIP suggesting that cervical sympathectomy may ameliorate neuroinflammation in the nervous system after SAH. In an experimental study, it was reported that cervical sympathectomy decreased neurogenic vasodilation in cranial dura mater in rats (Wei et al., 2011). Our findings are in concordance with this study. On the other hand, a previous study reported that SAH decreased plasma CGRP concentrations, whereas the blockade of sympathetic ganglion following SAH increased it in the experimental SAH model in rats (Hu et al., 2014). The plasma CGRP findings in our study were completely contrary to that ones in the study, but one 6
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therefore they may be diluted in total brain tissue. Moreover, main source of CGRP, SP and VIP are mostly cell bodies of neurons in trigeminal ganglion. Another possible explanation may be that CGRP, SP and VIP in the brain tissue may be expeditiously broken down or removed. Moreover, our findings revealed that the sympathectomy improved the neurological symptoms impaired by SAH. Someone can ask how did the sympathectomy improve the neurological symptoms without changing CGRP, SP and VIP concentrations in the brain tissue. In addition to above mentioned possible reasons may be valid for this, the sympathectomy may have protective effect against brain injury by attenuating oxidative and nitrative stress products and pro-inflammatory cytokines in the brain tissue as a result of prevention of cerebral vasospasm. On the other hand, the main finding of our study is that cervical sympathectomy following SAH reduced the SAH-induced c-fos expression in TNC, and levels of neuropeptides in the plasma, trigeminal ganglion and brainstem. This finding suggests that cervical sympathectomy treatment may be a promising therapeutic approach in the treatment of neuroinflammation in surviving patients with SAH. The current treatments for SAH contain hemodynamic augmentation and medically or surgically performed intra-arterial vasodilation (van Gijn and Rinkel, 2001). But, these approaches provide relatively less effect on the delayed neurological deteriorations. We can speculate that after such treatments, basal and evoked sympathetic discharges will still continue unless cervical sympathetic ganglion are blocked. This is a massive risk factor for surviving patients. Because SAH-induced local tissue mediators plus a small sympathetic stimulation could lead to nearly closure of cerebral and meningeal arteries. It was reported in clinical trials that blockade of different sympathetic ganglions can be carried out for the treatment of various conditions such as complex regional pain syndrome type I (reflex sympathetic dystrophy), causalgia, cluster or vascular headache, phantom limb pain, post-stroke pain, refractory angina pectoris, hyperhidrosis, Raynaud's syndrome, idiopathic livedo reticularis etc. (Mailis and Furlan, 2010; Bandyk et al., 2002; Ebrahim, 2011; Lowell et al., 1993). Sympathetic blockade can be performed in two different ways including chemical and surgical sympathectomy. While chemical sympathectomy has temporary therapeutic effects due to over time degradation of injected chemical agents, the effects of surgical sympathectomy is permanent and safer. Blockade of different sympathetic ganglions (like stellate ganglion) exhibits therapeutic effects by improving the blood supply of the region, and reducing plasma concentration of adrenal hormones. Above mentioned reports from the clinical trials suggested that surgical sympathectomy has therapeutic effects for the treatment of those painful conditions, and it exhibits unimportant side effects. Thus, cervical sympathectomy treatment may prevent early brain injury due to enhanced neurogenic inflammatory neuropeptides such as CGRP, SP and VIP, and consequently deficits in patients with SAH who were arrived to the hospital before occurrence of early brain injury. Here, it can be inquired whether sympathetic denervation of cerebral arteries as a result of the sympathectomy will adversely affect cerebral circulation. However, since the vasomotion of cerebral arteries/arterioles after sympathetic blockage can be mediated by mediators including local tissue factors, there may probably not be any disruption in the cerebral circulation in the long term. Furthermore, the sympathectomy may improve the life quality of patients by suppressing neurogenic inflammatory reactions in patients with SAH, who are late to hospital or survived after SAH. Therefore, surgical cervical sympathectomy treatment may be a good physiological treatment approach as an alternative to drug therapy after SAH.
However, cervical sympathectomy treatment reduced SAH-induced the increases in CGRP, SP and VIP levels in trigeminal ganglion and CGRP and SP levels in the brainstem. These findings suggest that the sympathectomy treatment shortly after SAH may prevent neurogenic inflammation at origin site of the inflammation. The superior cervical ganglion supplies sympathetic innervation to cerebral and meningeal arteries (Lv et al., 2014). There is a basal sympathetic nerve discharge to these vessels by cervical ganglion to maintain basal tonus of the vessels even if there is not any stimulus. Together basal tonus of the vessels due to basal sympathetic discharge plus vasospasm following SAH can lead to delayed brain deterioration, delayed cerebral ischemia, delayed neurological deficits and chronic head pain after SAH. Moreover a sympathetic activation over basal level after SAH could further promote cerebral vasospasm and correspondingly more neurological deteriorations could occur. In an experimental study, it was shown that vasoconstriction in middle and basal cerebral arteries occured following induction of experimental SAH in rats, however, this vasospasm was reduced by cervical sympathectomy application (Hu et al., 2014). However, (neuronal) cell body response approach including the changes in expression of certain neuropeptides in peripheral neurons after the axotomy may explain possible mechanism underlying the effect of cervical sympathectomy treatment after SAH in the present study. It is well established that chemical or surgical sympathectomy (axotomy) results in changes in gene expression of certain neuropeptides in peripheral neurons in vivo (Zigmond, 2012). These changes contain increases in expression of certain neuropeptides and decreases in some other neuropeptides (Zigmond, 2012). However, the changes for a neuropeptide can include either increase or decrease in different neural tissues. For instance, while SP increases in sympathetic and motor neurons following axotomy it reduces in sensory ganglia (HyattSachs et al., 1996). In addition, Zigmond’s group reported that chemical sympathectomy and postganglionic axotomy lead to increases in VIP mRNA levels in the rat superior cervical ganglia (Hyatt-Sachs et al., 1996; Mohney et al., 1994). Moreover they demonstrated that axotomy of sensory neurons in lumbar dorsal root ganglia reduced protein and mRNA levels of neuropeptides CGRP and SP in this ganglia (Shadiack et al., 2001). On the other hand, it was previously shown that axonal sprouts contain CGRP and SP in the dorsal root ganglia after sciatic nerve transection in rats (McLachlan and Hu, 1998). In the present study, we measured the concentrations of the neuropeptides in plasma and different tissues including trigeminal ganglia, brainstem and brain. Thus, in the present study, it can be queried whether cervical sympathectomy treatment after SAH may have decreased levels of neurogenic inflammatory neuropeptides CGRP, SP and VIP through the cell body response. Since we surgically excised superior cervical ganglia that contain the cell bodies of neurons contrary to the axons, it seems unlikely that cervical sympathectomy act as directly via this mechanism. However it may acts as indirectly via an unknown mechanism on the levels of these neuropeptides. Another question may be whether the changes occured in SAH plus sympathectomy can also happen in only sympathectomy if sympathectomy alone was carried out. It would be favorable if it was carried out also sympathectomy alone but this does not adversely affect our current results. Because sympathectomy treatment in current study didn’t include the axotomy and we used both naive and saline controls. However, in here we first showed that cervical sympathectomy alleviates SAH-induced neurogenic inflammatory neuropeptides CGRP, SP and VIP in the plasma, trigeminal ganglion and brainstem. Therefore, we believe that our findings can be able to pioneer further comprehensive studies on the effects of sympathectomy treatment following SAH. In addition, in the present study, neither SAH nor the sympathectomy had any effect on the CGRP, SP and VIP levels in total brain tissue. The most likely reason for this may be that the concentrations of these neuropeptides were measured from total brain tissue and
3.1. Limitations of the study In addition to immunohistochemical staining method that we used in the present study to determine c-fos positive-cells in TNC in the brain stem, it would be favorable if it was carried out also quantitative 7
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tilted caudally at a 30° angle over 20 s. The rats in the saline group were injected with equal volume of sterile saline in the same way. The burr hole was sealed with bone wax. The rats were injected (i.p.) with 1 ml of saline to prevent dehydration (i.p.) and then they were placed in the cages with a 30° head-down position, therefore, blood was provided to distribute around the basal arteries for 30 min. Two rats in the SAH + Symp group that died during surgical operations were excluded from the experiments.
Western blot or ELISA technique. Moreover if it was carried out also sympathectomy group alone, it would have further enriched current findings. 4. Materials and methods 4.1. Animals The animals used in the study were treated according to the Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011, The National Academies Press). Male Wistar rats weighing 200–250 g were used in the experiments. Rats were purchased from Abant Izzet Baysal University, the experimental animals implementation and research centre (Turkey). The rats were fed ad libitum with rodent food and water, and were housed in their individual cages at a room temperature (22 ± 2 °C) and a 12 h light/dark cycle. All experimental procedures were approved by Abant Izzet Baysal University, Local Ethics Committee for Animal Experiments (license number 2016/28).
5.2. Establishment of the TCSG model
Male Wistar rats (10 weeks old) were randomly divided into 4 groups: the control group (n = 7), no procedure was performed; Saline group (n = 14) was injected with 300 μl of normal saline into the prechiasmatic cistern; SAH + Sham group (n = 14) was exposed to induction of subarachnoid hemorrhage; SAH + Symp group (n = 14) was exposed to left transection of the cervical sympathetic ganglion following SAH induction. Moreover, for c-fos immunohistochemical staining, seven rats were used for each group. Experimental groups and surgical interventions are shown in Table 1.
Transection of the cervical sympathetic ganglion (TCSG) was carried out in rats in SAH + Symp group. One hour after induction of SAH, the left superior cervical sympathetic ganglion was removed as previously described by Kilinc et al. (Kilinc et al, 2015). Under still ketamine and xylazine anaesthesia (above mentioned) and under a dissecting microscope, a 1-cm incision was made aseptically in the ventral area of the neck, left sternocleidomastoid muscle was exposed and deflected, then the bifurcation of the left carotid artery were seen. Because of the left superior cervical ganglion is placed under bifurcation of the left carotid artery, the left carotid artery was displaced laterally to expose the superior cervical ganglion. Then, the superior cervical ganglion with pre- and post-ganglionic fibers was cut and removed via a fine-scissors and forceps, respectively. Adequate care was endeavoured not to damage the adjacent vagal and hypoglossal nerves. The incision was sutured under aseptic conditions. Success of transection of the cervical sympathetic ganglion was confirmed by ipsilateral ptosis (droopy eyelid). Left cervical sympathetic ganglions in the Saline and SAH+Symp groups were exposed but not transected. After surgery interventions, rats in all groups were kept alive for 14 days under appropriate conditions.
5. Materials
5.3. Neurological evaluation
Paraformaldehyde and phosphate-buffered saline were purchased from Sigma-Aldrich, (Schnelldorf, Germany), c-fos kit was purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA), CGRP, SP and VIP ELISA kits were purchased from ELABscience (Wuhan, P.R. China).
Neurological functions of the rats were assessed by a blinded investigator at 2th, 8th and 14th days following induction of SAH as previous described (Ersahin et al., 2009). In summary, a 20-point neurological score was used to evaluate motor and behavioral deficits. The motor and behavioral performance were graded in a score range of 0–20. Higher scores indicate severe neurological deficits.
4.2. Experimental groups
5.1. Induction of experimental SAH The experimental SAH model was constructed according to the procedure described in a previous study, which included the injection of autologous blood into the prechiasmatic cistern (Prunell et al., 2002). Briefly, after the rats were anesthetized with the intraperitoneal (ip) injection of ketamine (90 mg/kg, Ketalar; Pfizer, New York, NY) and xylazine (5 mg/kg; Rompun; Bayer, Leverkusen, Germany), they were placed prone in a stereotactic apparatus (Stoelting Europe, Dublin, Ireland). In aseptic conditions, a small incision was made in the midline to the scalp and a 1 mm hole was drilled in the skull 7.5 mm anterior to the bregma in the midline without damaging the dura mater. Then, 300 μl of autologous blood from the femoral artery was injected gently into the prechiasmatic cistern through a 26 G spinal needle which was
5.4. Blood collection, harvesting and homogenization of the tissues 14 days after surgical interventions, under anesthesia with ketamine (90 mg/kg, i.p.) thorax of rats was opened by a scissor, and blood samples were taken from the right ventricle by cardiac puncture. Rats, excluding used for c-fos immun-staining, were perfused intracardially with only 100 ml PBS (phosphate-buffered saline, pH: 7.4) to remove blood from structures of the nervous system. The head was separated from the body at the atlanto-occipital joint, the brain was removed. Brainstem including trigeminal nucleus caudalis area was dissected within the following limits: coronal sections from −9 to −15 mm posterior to bregma, 4 mm lateral to bregma (Paxinos’ the rat brain
Table 1 Experimental groups and surgical interventions. Group
Surgical interventions
Final number of animals
Control Saline
not touched (to determine naive concentrations of c-fos, CGRP, SP and VIP) 300 μl of normal saline was injected into the prechiasmatic cistern. One hour after saline injection, left cervical sympathetic ganglion were exposed but not transected 300 μl of autologous blood was injected into the prechiasmatic cistern. One hour after blood injection, left cervical sympathetic ganglion were exposed but not transected 300 μl of autologous blood was injected into the prechiasmatic cistern. One hour after blood injection, left superior cervical sympathetic ganglion was removed
7 14
SAH + Sham SAH + Symp
SAH: subarachnoid hemorrhage; Symp: sympathectomy. 8
14 14
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mean ± standard error of the mean. Statistical analysis was performed using SPSS for Windows (version 17.0, SPSS Inc., Chicago, IL, USA). The statistical differences between the different groups were analyzed by one-way analysis of variance (ANOVA) and followed by LSD test for multiple comparisons. A value of p < 0.05 was considered statistically significant.
atlas), with a depth of 2–3 mm. Trigeminal ganglia were dissected by cutting the nerve divisions approximately 2 mm proximal and distal to the branching point of the mandibular nerve. The dura was intently removed from the ganglia under stereo-microscope. The brain, brainstem and trigeminal ganglia samples were put into the tubes, and the tubes were immediately placed in liquid nitrogen. Blood samples were centrifuged at 3000 × g for 15 min at 4 °C. Homogenization of brain tissue, brainstem and trigeminal ganglia samples was performed using a light-duty Ultra-Turrax homogenizer (ISOLAB, Wertheim, Germany) as previously described by Kilinc et al. (2018). The supernatants were stored at −80 °C until assayed for CGRP, SP and VIP immunoreactivities by ELISA. On the other hand, rats to be used for immunohistochemical staining for c-fos expression were perfused intracardially with 100 ml PBS (phosphate-buffered saline, pH: 7.4) and followed by 200 ml 4% paraformaldehyde (PAF). Brainstem samples were incubated overnight in 4% PAF.
Acknowledgements We declare that there are not any relationships that present a potential conflict of interest. We thank Shawnda Camsari who carried out the proofreading of this article in terms of English grammar. Funding This study was supported by Abant Izzet Baysal University Scientific Research Fund (Grant number: 2016.08.09.1096).
5.5. Determination of CGRP, SP and VIP concentrations in plasma and the tissues
References
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