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Changes of PACAP level in cerebrospinal fluid and plasma of patients with severe traumatic brain injury
Peptides 60 (2014) 18–22
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Changes of PACAP level in cerebrospinal fluid and plasma of patients with severe traumatic brain injury Peter Bukovics a,b,1 , Endre Czeiter b,c,d,e,∗,1 , Krisztina Amrein a , Noemi Kovacs a , Jozsef Pal a , Andrea Tamas d,e, Terez Bagoly f, Zsuzsanna Helyes c,f, Andras Buki a,b,c,1, Dora Reglodi d,e,1 a
Department of Neurosurgery, University of Pecs, Pecs, Hungary MTA-PTE Clinical Neuroscience MR Research Group, Pecs, Hungary c Janos Szentagothai Research Centre, University of Pecs, Pecs, Hungary d MTA-PTE Lendulet PACAP Research Group, Pecs, Hungary e Department of Anatomy, University of Pecs, Pecs, Hungary f Department of Pharmacology and Pharmacotherapy, University of Pecs, Pecs, Hungary b
a r t i c l e
i n f o
Article history: Received 22 April 2014 Received in revised form 1 July 2014 Accepted 1 July 2014 Available online 10 July 2014 Keywords: Endogenous PACAP Plasma Cerebrospinal fluid Traumatic brain injury Neuroprotective Blood–brain barrier
a b s t r a c t PACAP has well-known neuroprotective potential including traumatic brain injury (TBI). Its level is up-regulated following various insults of the CNS in animal models. A few studies have documented alterations of PACAP levels in human serum. The time course of post-ictal PACAP levels, for example, show correlation with migraine severity. Very little is known about the course of PACAP levels following CNS injury in humans and the presence of PACAP has not yet been detected in cerebrospinal fluid (CSF) of subjects with severe TBI (sTBI). The aim of the present study was to determine whether PACAP occurs in the CSF and plasma (Pl) of patients that suffered sTBI and to establish a time course of PACAP levels in the CSF and Pl. Thirty eight subjects with sTBI were enrolled with a Glasgow Coma Scale ≤8 on admission. Samples were taken daily, until the time of death or for maximum 10 days. Our results demonstrated that PACAP was detectable in the CSF, with higher concentrations in patients with TBI. PACAP concentrations markedly increased in both Pl and CSF in the majority of patients 24–48 h after the injury stayed high thereafter. In cases of surviving patients, Pl and CSF levels displayed parallel patterns, which may imply the damage of the blood–brain barrier. However, in patients, who died within the first week, Pl levels were markedly higher than CSF levels, possibly indicating the prognostic value of high Pl PACAP levels. © 2014 Elsevier Inc. All rights reserved.
Introduction Severe traumatic brain injury (sTBI) is one of the major causes of death among young individuals in high income countries [23]. According to the WHO’s estimation, traumatic brain injury will be the third most frequent cause of death until 2020 all over the world [28]. Neurotrophic factors are important endogenous neuroprotectants that play a role in spontaneous recovery following different types of neuronal injuries and are promising therapeutic tools [5,17–19,37]. Multiple lines of recently collected evidences point to the neuroprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP). This member of the vasoactive intestinal peptide (VIP)/secretin/glucagon peptide family was
∗ Corresponding author. Tel.: +36 20 557 6026. E-mail address: [email protected] (E. Czeiter). 1 P. Bukovics and E. Czeiter as well as A. Buki and D. Reglodi contributed equally to the present work. http://dx.doi.org/10.1016/j.peptides.2014.07.001 0196-9781/© 2014 Elsevier Inc. All rights reserved.
discovered as a hypothalamic peptide based on its potential to increase adenylate cyclase activity in the pituitary gland [27]. It is found in two forms, PACAP-27 and PACAP-38, with PACAP-38 being the major form [49]. PACAP is widely distributed in the nervous system and also in endocrine glands, cardiovascular, gastrointestinal and respiratory tracts. It is involved in the regulation of various physiological processes, such as feeding, reproduction, thermoregulation, catecholamine synthesis, motor activity, brain development and neuronal survival. PACAP has potent neurotrophic and neuroprotective actions in models of cerebral ischemia, Parkinson’s disease and retinal degeneration [2,3,5,30–35,38,39,42,45]. In spite of the similarities in the pathogenesis of ischemic and traumatic brain injuries [7], until the most recent years, only a few of studies have investigated the potential neuroprotective role of PACAP in traumatic brain or spinal cord injuries. In the extradural static weight compression model of spinal cord injury in rat, post-injury PACAP treatment significantly reduced the number of apoptotic cells in the injured spinal cord [8,21]. PACAP has also been shown to attenuate
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the increase of tumor necrosis factor after spinal cord transection [22]. In a focal traumatic brain injury (TBI) model, upregulation of PACAP mRNA was observed in parallel with decreased number of apoptotic cells [41]. Not only these studies primarily conducted in focal models of brain injury suggest that PACAP might be a promising tool to inhibit traumatic injuries of the central nervous system, but also our observations have demonstrated that PACAP successfully interfered with traumatically evoked diffuse axonal injury. In the last few years our laboratories extensively investigated the purported neuroprotective role of PACAP in a diffuse model of experimental TBI. First, we discovered that the administration paradigm proved successful in experimental stroke (125 g pre-injury, iv) did not exert beneficial effects in terms of inhibiting traumatically evoked axonal injury; however, intracerebroventricular (icv) injection of the same dose revealed significant axonoprotection [16,31]. We also established the dose-response curve for icv administration of PACAP, demonstrating that 100 g PACAP significantly reduced the density of damaged axons in the corticospinal tract. Further, our most recent observations indicate that a considerable therapeutic window exists for post-injury PACAP-treatment in diffuse experimental TBI, considering that significant axonoprotection could be observed when PACAP was administered 2 h post-injury [46]. On the basis of the above detailed observations PACAP is now considered as a potential candidate for clinical studies in TBI. In a PACAP-deficient mouse model of neuronal injury Armstrong and colleagues found that although motor neuron survival after axotomy was not significantly different in PACAP deficient vs. wild type mice, recovery of axon regeneration after crush injury was significantly delayed. This observation raises the novel hypothesis that endogenous PACAP is critically involved in a carefully controlled immune response that is necessary for proper nerve regeneration after injury [1]. Little is known about the functions of PACAP in humans. The presence of PACAP has been shown in rat and human blood. Radioimmunoassay (RIA) analysis of rat serum has revealed that PACAP-38 concentration is higher in the hypophyseal portal blood than in the peripheral circulation [15]. We have previously identified PACAP-38 in the rat plasma by mass spectrometry and provided evidence that PACAP is released from activated capsaicin-sensitive afferents into the circulation [20,29]. In light of the above the aims of our present study were (1) to determine the presence of PACAP in the cerebrospinal fluid (CSF) and the plasma of patients with severe TBI, (2) to compare PACAP levels in sTBI with controls and to gain an initial insight into the time course of PACAP levels after sTBI and (3) to implement pilot statistical analysis whether there is any significant correlation between the PACAP levels and the clinical outcome.
Patients and methods Study population characteristics Human blood and CSF samples were taken daily from 38 patients who suffered severe TBI and were admitted to Department of Neurosurgery, University of Pecs, Hungary. The main inclusion criterion was Glasgow Coma Scale (GCS) ≤8 on admission caused by sustained severe TBI less than 24 h before the enrollment. Exclusion criteria were age <18, known autoimmune disease, and pregnancy. Because of the comatose state of these patients informed consents were obtained from legally authorized representatives. The patient enrollment, sample collection, and management were carried out with the permission of the local Institutional Review Board (IRB) rigorously complied with the relating rules of the Hungarian law and the Good Clinical Practice.
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Outcome of the patients was defined as survived or not survived of the first week post-injury. Sample collection The first samples were collected at the time of enrollment and then daily until the time of death or for a maximum duration of 10 days. Samples were only obtained by trained individuals using aseptic techniques and extreme care. For taking plasma purple top tubes (K2 EDTA), for collecting CSF red top tubes (plain) were used. Tubes were sat at room temperature for 5–10 min, and the samples were then centrifuged at 4000 rpm for 8 min to gain the plasma and clear CSF. Plasma and CSF were apportioned into 1.8 ml cryovials (aliquot). Fourteen control plasma samples were collected from healthy volunteers with the previously described manner. Our 8 control CSF samples derived from lumbar puncture of patients who were punctured with suspected neurological diseases (e.g.: meningitis) but found negative. PACAP-38 level measurement PACAP-38-like immunoreactivity in the human plasma and CSF was determined with a specific and sensitive RIA technique developed in our laboratory [20,29] and concentrations of the peptide were calculated with a calibration curve. The peptide was extracted from 3 ml plasma samples by addition of a double volume of absolute alcohol and 20 l 96% acetic acid. After precipitation and a second centrifugation (3000 rpm for 20 min at 4 ◦ C) the samples were dried under nitrogen flow and resuspended in 300 l assay buffer to achieve a 10 times higher concentration for the RIA procedure [20,29]. For RIA analysis, 10 l 96% acetic acid was added to 1 ml sample and incubated in 40 ◦ C water bath for 5 min to precipitate the protein content. RIA analysis using the “881113” PACAP-38 antiserum was performed as previously described [20,29]. PACAP-38 concentrations of the samples were read from a calibration curve created using the standard preparations. For statistical purposes we determined the following PACAP-38 levels in the CSF and plasma samples of each patient: first CSF and plasma level, CSF and plasma levels in the second post-traumatic day, highest CSF and plasma levels, average of all CSF and plasma levels. Statistical analysis All measured PACAP levels are in fmol/ml ± SD. To compare plasma and CSF levels of PACAP-38 of the patients with the healthy controls we utilized one way ANOVA with Scheffé post hoc test. In attempt to find correlation between the previously described PACAP-38 levels and the first week mortality we utilized univariate logistic regression analysis. All statistical procedures were carried out by IBM SPSS Statistics 22 for Windows software. Statistical results were considered significant when p < 0.05. Results We enrolled 38 subjects with sustained sTBI into the current study. The age range was between 18 and 83 years with a mean age of 57.97 ± 17.70 years (11 females and 27 males). The on admission GCS was 4.78 ± 1.64. The descriptive statistical analysis of our raw data already showed that all the CSF and plasma PACAP levels were markedly elevated compared to the normal controls although we could observe a highly increased standard deviation at the same time. Table 1 summarizes the results of the descriptive statistical analysis.
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Table 1 Descriptive statistical parameters of PACAP-38 levels. (All PACAP-38 levels are expressed in fmol/ml.).
First CSF Mean CSF Highest CSF Day2 CSF Control CSF First Pl Mean Pl Highest Pl Day2 Pl Control Pl
n
Mean
Median
SD
Minimum
Maximum
26 34 34 25 8 35 38 38 30 14
25.262 28.978 39.677 32.363 16.090 30.263 37.458 51.401 38.788 14.514
24.700 28.965 36.850 32.300 16.067 28.100 36.461 51.000 34.862 14.650
14.134 10.116 19.421 14.220 1.018 13.243 12.463 16.564 18.793 3.080
5.000 8.326 14.000 10.800 14.600 10.700 13.600 19.700 14.400 10.700
79.400 46.645 83.031 83.031 17.450 68.200 68.325 92.800 89.500 21.000
Table 2 Results of univariate logistic regression to the first week mortality. Significant connections revealed with the mean plasma and day2 plasma concentrations. (OR = odds ratio; CI = confidence interval; R2 = Nagelkerke R2 levels.).
First CSF Mean CSF Highest CSF Day 2 CSF First plasma Mean plasma Highest plasma Day 2 plasma
OR (95% CI)
p
R2
1.028 (0.970–1.090) 0.980 (0.905–1.061) 0.999 (0.958–1.041) 1.015 (0.952–1.084) 1.032 (0.977–1.090) 1.143 (1.032–1.267) 1.039 (0.991–1.088) 1.057 (1.003–1.114)
0.348 0.614 0.946 0.643 0.262 0.010 0.110 0.037
0.040 0.011 0.000 0.013 0.048 0.403 0.102 0.256
Discussion For the comparison of sTBI and control PACAP levels in CSF and Pl we utilized one way ANOVA and found significant differences p = 8.45 × 10−13 in case of Pl and p = 6.33 × 10−5 in case of CSF. The Scheffé post hoc test revealed significant difference between control CSF and highest CSF levels and also significant differences were proven between control Pl levels and the first, mean, highest and day 2 plasma levels (Fig. 1). Univariate logistic regression analysis revealed significant connection between the mean and the second post-injury day plasma PACAP levels and the first week mortality (Table 2). To gain further insight into the connection between PACAP levels and the outcome we investigated the relationship between the time course of CSF and plasma PACAP levels and the outcome (Fig. 2). Although we were not able to gain statistically significant difference a clear tendency was well observable that the average plasma levels of the subjects who died within the first post injury week were almost doubled during the entire time period compared to the CSF levels of the same subjects and the CSF and plasma levels of those who survived.
Fig. 1. CSF (A) and Plasma (B) levels of PACAP compared to the controls. Asterisks show the revealed significant difference between highest CSF levels and the controls. In case of plasma concentrations all sTBI levels showed significant difference from the controls. (All measured levels are in fmol/ml.)
In our present study we confirmed that PACAP existed both in plasma and CSF of severe TBI patients as well as non-head injured human controls. Elevated plasma and CSF levels were found in case of severe head injury compared to the controls in each biofluid. Although we were able to find significant connection between plasma PACAP levels and the first week mortality we failed to establish any relationship with clinical parameters. As far as the time course of PACAP levels are concerned, we detected a clear tendency to have a higher plasma PACAP level in case of subjects who died within the first week. Although our results raise several different questions, probably the most important one is the source of the extra amount of PACAP in the biofluids of the sTBI patients compared to the normal controls during the entire investigational period. There are many different pathomechanisms – or a combination of them – which may play a part in the background of this finding.
Fig. 2. Time courses of PACAP38 plasma and CSF levels of all the subjects during the ten days post-injury period (A) and time courses of the plasma and CSF levels of PACAP38 during the first five days post-injury—the lethal cases during the first week compared to the survivors (B). (All measured levels are in fmol/ml.)
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From a simple mechanistical point of view it may seem elegant to account the elevated PACAP levels in the sTBI patients’ biofluids to the primary tissue loss at the time point of the injury. This theory is also supported by the fact that significant decrease of PACAP38 immunoreactivity was found in the primary lesion site in human pathologic samples [47]. However, considering the fact that the estimated half-life of PACAP38 in the human blood is not more than 5–10 min [24,48], only secondary injuries can be responsible for the observed prolonged elevation. As a number of different studies have confirmed the neuroprotective effect of PACAP [2,30,31,33,38,42,43], it seemed reasonable to explain the elevated PACAP levels of sTBI subjects with an endogenous overproduction of PACAP as a pathophysiological response to the loss of neural tissue in the CNS. Landeghem et al. described the cellular localization of PACAP in human brain after TBI. They found a prolonged decrease in PACAP38 content in the lesion site, while a significant increase of PACAP38 positive cells was detected in the penumbra region [47]. A substantially weak point of this concept is that it does not efficiently explain the markedly high concentrations of PACAP in the plasma of the subjects – especially in the lethal cases – during the first post-injury week. In the background of the elevated plasma PACAP levels the damage of the blood–brain barrier (BBB) may be suspected, which – as it is widely described – is very commonly associated with sTBI [9,36,40]. Moreover, the extent of BBB disruption can be quantified based upon the quotient of CSF and serum albumin concentration and biomarker molecules like S100 protein [6]. This logic concept has at least two limitations: (1) although it probably plays a significant role in the increase of PACAP in the peripheral blood, it simply physically cannot be responsible for the even higher PACAP concentrations in the plasma then in CSF—as this phenomenon is clearly visible in Fig. 2B. (2) While PACAP38 has its own long described transport mechanisms even through the intact BBB [4], results from our lab previously showed that its intravenous administration was ineffective in contrast to an icv administration—in an experimental setting of sTBI [16]. As systemic inflammatory response is a quite common complication associated with sTBI [10], frequently even leading to systemic inflammatory response syndrome (SIRS) [25]. PACAP has a well described anti-inflammatory potential [12,13,26]—the elevated PACAP38 levels in sTBI patients biofluids can be explained by an endogenous response to the exaggerated immune reaction. Furthermore, this theory supports the tendency of markedly higher plasma levels in the lethal cases probably due to a more severe systemic inflammatory response than in the survivors. An interesting side stream of the considerations regarding the possible pathomechanisms behind the elevated plasma PACAP38 levels is the potential role of ceruloplasmin. Ceruloplasmin was identified as the binding factor of PACAP38 in the human plasma [44] and the level of ceruloplasmin was also associated with TBI as a potential biomarker [11]. Relatively low levels of ceruloplasmin were described as a predictor of intracranial pressure elevations—it cannot be ruled out that the low ceruloplasmin levels controversially lead to more elevated plasma PACAP38 concentrations in the more severe (lethal) cases. The fact that a significant connection was found between plasma PACAP38 levels and the first week mortality supports PACAP’s potential role as a biomarker. However, it should be emphasized that as only the plasma concentration of the second post-injury day and the average plasma values were significantly predictive, PACAP appears much less effective than the current best set of protein biomarker candidates of TBI like GFAP or UCH-L1 [14]. Nevertheless, our results still draw the attention to the necessity of further more precisely focused investigations regarding PACAP38 as a potential biomarker in large cohorts of subjects. First of
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all – compared to the above described short half-life of PACAP with our hereby utilized sampling protocol – the applicability of PACAP38 as an acute biomarker of the primary tissue loss seems worth to be tested by studies which involve blood sampling at the injury scene (by the ambulance). Further clarification of the role and/or cause of the elevated blood and CSF PACAP38 levels in the pathophysiology of sTBI may reveal that PACAP could be a biomarker of a subprocess like extent of the BBB disruption, initiation of endogenous neuroprotective processes or the state of the systemic inflammatory response. We must draw the attention to substantial limiting factors that may affect our presented results. The number of patients as well as the number of centers could be increased along with a more frequent sampling. This protocol would increase the data available to ensure a more detailed insight into the time course of a molecule with rapid turnover in body fluids. In spite of its shortcomings, we believe that our study contributes to a better understanding of the possible role(s) of PACAP in human sTBI and could serve as a good source for multi-center clinical trials which involve this topic. Conclusions To the best of our knowledge this is the first study where the presence of PACAP and its level in the plasma and in the CSF was measured in TBI patients; our results represent the first publication of the finding that PACAP should be detectable both in the plasma and in the CSF of the severely head injured. Both the plasma and the highest CSF levels showed significant increase compared to the controls. Although our results considered preliminary and limited by many factors (relatively small number of cases, single site) we were able to prove significant connection between plasma PACAP levels and the first week mortality. In our opinion the hereby presented data underlines the necessity of further investigations regarding the potential beneficial or deleterious effects of increased PACAP CSF and blood levels after severe head injury in the near future. Conflicts of interest statement The authors hereby declare that no competing financial interests exist. Acknowledgements This study was supported by National Science Research Found OTKA PD72240, OTKA K104984, the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of SROP 4.2.4.A/2-11-1-2012-0001, SROP-4.2.2.A-11/1/ KONV-2012-0017, SROP-4.2.2.A-11/1/KONV-2012-0024; ‘National Excellence Program’; Magyary Zoltan Scholarship; Arimura Foundation; MTA-PTE “Lendület” Program and Hungarian Brain Research Program—Grant No. KTIA 13 NAP-A-III/5 as well as Hungarian Brain Research Program—Grant No. KTIA 13 NAP-A-II/8. References [1] Armstrong B, Abad C, Chhith S, Cheung-Lau G, Hajji O, Nobuta H, et al. Impaired nerve regeneration and enhanced neuroinflammatory response in mice lacking pituitary adenylyl cyclase activating peptide. Neuroscience 2008;151:63–73. [2] Atlasz T, Babai N, Kiss P, Reglodi D, Tamás A, Szabadfi K, et al. Pituitary adenylate cyclase activating polypeptide is protective in bilateral carotid occlusioninduced retinal lesion in rats. Gen Comp Endocrinol 2007;153:108–14. [3] Babai N, Atlasz T, Tamás A, Reglödi D, Tóth G, Kiss P, et al. Degree of damage compensation by various PACAP treatments in monosodium glutamate-induced retinal degeneration. Neurotox Res 2005;8:227–33. [4] Banks WA, Kastin AJ, Komaki G, Arimura A. Passage of pituitary adenylate cyclase activating polypeptide1-27 and pituitary adenylate cyclase activating polypeptide1-38 across the blood–brain barrier. J Pharmacol Exp Ther 1993;267:690–6.
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[5] Beal M, Palomo T, Kostrzewa R, Archer T. Neuroprotective and neurorestorative strategies for neuronal injury. Neurotox Res 2000;2:71–84. [6] Blyth BJ, Farhavar A, Gee C, Hawthorn B, He H, Nayak A, et al. Validation of serum markers for blood–brain barrier disruption in traumatic brain injury. J Neurotrauma 2009;26:1497–507. [7] Bramlett H, Dietrich W. Pathophysiology of cerebral ischemia and brain trauma: similarities and differences. J Cereb Blood Flow Metab 2004;24:133–50. [8] Chen W, Tzeng S. Pituitary adenylate cyclase-activating polypeptide prevents cell death in the spinal cord with traumatic injury. Neurosci Lett 2005;384:117–21. [9] Chodobski A, Zink BJ, Szmydynger-Chodobska J. Blood–brain barrier pathophysiology in traumatic brain injury. Transl Stroke Res 2011;2:492–516. [10] Das M, Mohapatra S, Mohapatra SS. New perspectives on central and peripheral immune responses to acute traumatic brain injury. J Neuroinflammation 2012;9:12. [11] Dash PK, Redell JB, Hergenroeder G, Zhao J, Clifton GL, Moore A. Serum ceruloplasmin and copper are early biomarkers for traumatic brain injury-associated elevated intracranial pressure. J Neurosci Res 2010;88:1719–26. [12] Delgado M, Abad C, Martinez C, Juarranz MG, Leceta J, Ganea D, et al. PACAP in immunity and inflammation. Ann N Y Acad Sci, 992; 2003. p. 141–57. [13] Delgado M, Martinez C, Pozo D, Calvo JR, Leceta J, Ganea D, et al. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activation polypeptide (PACAP) protect mice from lethal endotoxemia through the inhibition of TNFalpha and IL-6. J Immunol 1999;162:1200–5. [14] Diaz-Arrastia R, Wang KK, Papa L, Sorani MD, Yue JK, Puccio AM, et al. Acute biomarkers of traumatic brain injury: relationship between plasma levels of ubiquitin C-terminal hydrolase-L1 and glial fibrillary acidic protein. J Neurotrauma 2014;31:19–25. [15] Dow R, Bennie J, Fink G. Pituitary adenylate cyclase-activating peptide-38 (PACAP)-38 is released into hypophysial portal blood in the normal male and female rat. J Endocrinol 1994;142:R1–4. [16] Farkas O, Tamas A, Zsombok A, Reglodi D, Pal J, Buki A, et al. Effects of pituitary adenylate cyclase activating polypeptide in a rat model of traumatic brain injury. Regul Pept 2004;123:69–75. [17] Feng S, Kong X, Guo S, Wang P, Li L, Zhong J, et al. Treatment of spinal cord injury with co-grafts of genetically modified Schwann cells and fetal spinal cord cell suspension in the rat. Neurotox Res 2005;7:169–77. [18] Garcia de Yebenes J, Yebenes J, Mena M. Neurotrophic factors in neurodegenerative disorders: model of Parkinson’s disease. Neurotox Res 2000;2:115–37. [19] Gerlach M, Double K, Youdim M, Riederer P. Strategies for the protection of dopaminergic neurons against neurotoxicity. Neurotox Res 2000;2:99–114. [20] Helyes Z, Pozsgai G, Börzsei R, Németh J, Bagoly T, Márk L, et al. Inhibitory effect of PACAP-38 on acute neurogenic and non-neurogenic inflammatory processes in the rat. Peptides 2007;28:1847–55. [21] Katahira M, Yone K, Arishima Y, Nagamine T, Komiya S, Iwata S, et al. The neuroprotective effects of PACAP on spinal cord injury (SCI) in rats. Regul Pept 2003;115:49. [22] Kim W, Kan Y, Ganea D, Hart R, Gozes I, Jonakait G. Vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide inhibit tumor necrosis factor-alpha production in injured spinal cord and in activated microglia via a cAMP-dependent pathway. J Neurosci 2000;20:3622–30. [23] Langlois J, Rutland-Brown W, Thomas K. Traumatic brain injury in the United States: emergency department visits, hospitalizations, and deaths. Atlanta, GA: Centers for Disease Control and Prevention, National Center for Injury Prevention and Control; 2006. [24] Li M, Maderdrut JL, Lertora JJL, Batuman V. Intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP) in a patient with multiple myeloma and myeloma kidney: a case study. Peptides 2007;28:1891–5. [25] Lu J, Goh SJ, Tng PYL, Deng YY, Ling EA, Moochhala S. Systemic inflammatory response following acute traumatic brain injury. Front Biosci, Landmark 2009;14:3795–813. [26] Martinez C, Arranz A, Juarranz Y, Abad C, Garcia-Gomez M, Rosignoli F, et al. PAC1 receptor: emerging target for septic shock therapy. Ann N Y Acad Sci, 1070; 2006. p. 405–10. [27] Miyata A, Arimura A, Dahl R, Minamino N, Uehara A, Jiang L, et al. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 1989;164:567–74.
[28] Murray CJL, Lopez AD. Alternative projections of mortality and disability by cause 1990–2020: global burden of disease study. Lancet 1997;349:1498–504. [29] Németh J, Reglödi D, Pozsgai G, Szabó A, Elekes K, Pintér E, et al. Effect of pituitary adenylate cyclase activating polypeptide-38 on sensory neuropeptide release and neurogenic inflammation in rats and mice. Neuroscience 2006;143:223–30. [30] Racz B, Reglodi D, Kiss P, Babai N, Atlasz T, Gabriel R, et al. In vivo neuroprotection by PACAP in excitotoxic retinal injury: review of effects on retinal morphology and apoptotic signal transduction. Int J Neuroprot Neurodeg 2006;2:80–5. [31] Reglodi D, Tamas A, Lubics A, Szalontay L, Zsombok A, Farkas O, et al. Recent results on the neuroprotective effects of PACAP in rat models of focal cerebral ischemia, Parkinson’s disease and traumatic brain injury. Neurotox Res 2005;8:317. [32] Reglodi D, Tamás A, Lubics A, Szalontay L, Lengvári I. Morphological and functional effects of PACAP in 6-hydroxydopamine-induced lesion of the substantia nigra in rats. Regul Pept 2004;123:85–94. [33] Reglodi D, Tamás A, Somogyvári-Vigh A, Szántó Z, Kertes E, Lénárd L, et al. Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia. Peptides 2002;23: 2227–34. [34] Reglödi D, Lubics A, Kiss P, Lengvári I, Gaszner B, Tóth G, et al. Effect of PACAP in 6-OHDA-induced injury of the substantia nigra in intact young and ovariectomized female rats. Neuropeptides 2006;40:265–74. [35] Rácz B, Gallyas FJ, Kiss P, Tóth G, Hegyi O, Gasz B, et al. The neuroprotective effects of PACAP in monosodium glutamate-induced retinal lesion involve inhibition of proapoptotic signaling pathways. Regul Pept 2006;137: 20–6. [36] Saw MM, Chamberlain J, Barr M, Morgan MP, Burnett JR, Ho KM. Differential disruption of blood–brain barrier in severe traumatic brain injury. Neurocrit Care 2014;20:209–16. [37] Segura-Aguilar J, Kostrzewa R. Neurotoxins and neurotoxicity mechanisms. An overview. Neurotox Res 2006;10:263–87. [38] Shioda S, Ohtaki H, Nakamachi T, Dohi K, Watanabe J, Nakajo S, et al. Pleiotropic functions of PACAP in the CNS: neuroprotection and neurodevelopment. Ann NY Acad Sci 2006;1070:550–60. [39] Shioda S, Ozawa H, Dohi K, Mizushima H, Matsumoto K, Nakajo S, et al. PACAP protects hippocampal neurons against apoptosis: involvement of JNK/SAPK signaling pathway. Ann NY Acad Sci 1998;865:111–7. [40] Shlosberg D, Benifla M, Kaufer D, Friedman A. Blood–brain barrier breakdown as a therapeutic target in traumatic brain injury. Nat Rev Neurol 2010;6:393–403. [41] Skoglösa Y, Lewén A, Takei N, Hillered L, Lindholm D. Regulation of pituitary adenylate cyclase activating polypeptide and its receptor type 1 after traumatic brain injury: comparison with brain-derived neurotrophic factor and the induction of neuronal cell death. Neuroscience 1999;90:235–47. [42] Somogyvári-Vigh A, Reglodi D. Pituitary adenylate cyclase activating polypeptide: a potential neuroprotective peptide. Curr Pharm Des 2004;10:2861–89. [43] Tamas A, Reglodi D, Farkas O, Kovesdi E, Pal J, Povlishock JT, et al. Effect of PACAP in central and peripheral nerve injuries. Int J Mol Sci 2012;13:8430–48. [44] Tams JW, Johnsen AH, Fahrenkrug J. Identification of pituitary adenylate cyclase-activating polypeptide 1-38-binding factor in human plasma, as ceruloplasmin. Biochem J 1999;341:271–6. [45] Tamás A, Lubics A, Lengvári I, Reglódi D. Protective effects of PACAP in excitotoxic striatal lesion. Ann NY Acad Sci 2006;1070:570–4. [46] Tamás A, Zsombok A, Farkas O, Reglödi D, Pál J, Büki A, et al. Postinjury administration of pituitary adenylate cyclase activating polypeptide (PACAP) attenuates traumatically induced axonal injury in rats. J Neurotrauma 2006;23:686–95. [47] van Landeghem FKH, Weiss T, Oehmichen M, von Deimling A. Cellular localization of pituitary adenylate cyclase-activating peptide (PACAP) following traumatic brain injury in humans. Acta Neuropathol 2007;113:683–93. [48] Vaudry D, Falluel-Morel A, Bourgault S, Basille M, Burel D, Wurtz O, et al. Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery. Pharmacol Rev 2009;61:283–357. [49] Vaudry D, Gonzalez B, Basille M, Yon L, Fournier A, Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 2000;52:269–324.
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Changes of PACAP level in cerebrospinal fluid and plasma of patients with severe traumatic brain injury Peter Bukovics a,b,1 , Endre Czeiter b,c,d,e,∗,1 , Krisztina Amrein a , Noemi Kovacs a , Jozsef Pal a , Andrea Tamas d,e, Terez Bagoly f, Zsuzsanna Helyes c,f, Andras Buki a,b,c,1, Dora Reglodi d,e,1 a
Department of Neurosurgery, University of Pecs, Pecs, Hungary MTA-PTE Clinical Neuroscience MR Research Group, Pecs, Hungary c Janos Szentagothai Research Centre, University of Pecs, Pecs, Hungary d MTA-PTE Lendulet PACAP Research Group, Pecs, Hungary e Department of Anatomy, University of Pecs, Pecs, Hungary f Department of Pharmacology and Pharmacotherapy, University of Pecs, Pecs, Hungary b
a r t i c l e
i n f o
Article history: Received 22 April 2014 Received in revised form 1 July 2014 Accepted 1 July 2014 Available online 10 July 2014 Keywords: Endogenous PACAP Plasma Cerebrospinal fluid Traumatic brain injury Neuroprotective Blood–brain barrier
a b s t r a c t PACAP has well-known neuroprotective potential including traumatic brain injury (TBI). Its level is up-regulated following various insults of the CNS in animal models. A few studies have documented alterations of PACAP levels in human serum. The time course of post-ictal PACAP levels, for example, show correlation with migraine severity. Very little is known about the course of PACAP levels following CNS injury in humans and the presence of PACAP has not yet been detected in cerebrospinal fluid (CSF) of subjects with severe TBI (sTBI). The aim of the present study was to determine whether PACAP occurs in the CSF and plasma (Pl) of patients that suffered sTBI and to establish a time course of PACAP levels in the CSF and Pl. Thirty eight subjects with sTBI were enrolled with a Glasgow Coma Scale ≤8 on admission. Samples were taken daily, until the time of death or for maximum 10 days. Our results demonstrated that PACAP was detectable in the CSF, with higher concentrations in patients with TBI. PACAP concentrations markedly increased in both Pl and CSF in the majority of patients 24–48 h after the injury stayed high thereafter. In cases of surviving patients, Pl and CSF levels displayed parallel patterns, which may imply the damage of the blood–brain barrier. However, in patients, who died within the first week, Pl levels were markedly higher than CSF levels, possibly indicating the prognostic value of high Pl PACAP levels. © 2014 Elsevier Inc. All rights reserved.
Introduction Severe traumatic brain injury (sTBI) is one of the major causes of death among young individuals in high income countries [23]. According to the WHO’s estimation, traumatic brain injury will be the third most frequent cause of death until 2020 all over the world [28]. Neurotrophic factors are important endogenous neuroprotectants that play a role in spontaneous recovery following different types of neuronal injuries and are promising therapeutic tools [5,17–19,37]. Multiple lines of recently collected evidences point to the neuroprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP). This member of the vasoactive intestinal peptide (VIP)/secretin/glucagon peptide family was
∗ Corresponding author. Tel.: +36 20 557 6026. E-mail address: [email protected] (E. Czeiter). 1 P. Bukovics and E. Czeiter as well as A. Buki and D. Reglodi contributed equally to the present work. http://dx.doi.org/10.1016/j.peptides.2014.07.001 0196-9781/© 2014 Elsevier Inc. All rights reserved.
discovered as a hypothalamic peptide based on its potential to increase adenylate cyclase activity in the pituitary gland [27]. It is found in two forms, PACAP-27 and PACAP-38, with PACAP-38 being the major form [49]. PACAP is widely distributed in the nervous system and also in endocrine glands, cardiovascular, gastrointestinal and respiratory tracts. It is involved in the regulation of various physiological processes, such as feeding, reproduction, thermoregulation, catecholamine synthesis, motor activity, brain development and neuronal survival. PACAP has potent neurotrophic and neuroprotective actions in models of cerebral ischemia, Parkinson’s disease and retinal degeneration [2,3,5,30–35,38,39,42,45]. In spite of the similarities in the pathogenesis of ischemic and traumatic brain injuries [7], until the most recent years, only a few of studies have investigated the potential neuroprotective role of PACAP in traumatic brain or spinal cord injuries. In the extradural static weight compression model of spinal cord injury in rat, post-injury PACAP treatment significantly reduced the number of apoptotic cells in the injured spinal cord [8,21]. PACAP has also been shown to attenuate
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the increase of tumor necrosis factor after spinal cord transection [22]. In a focal traumatic brain injury (TBI) model, upregulation of PACAP mRNA was observed in parallel with decreased number of apoptotic cells [41]. Not only these studies primarily conducted in focal models of brain injury suggest that PACAP might be a promising tool to inhibit traumatic injuries of the central nervous system, but also our observations have demonstrated that PACAP successfully interfered with traumatically evoked diffuse axonal injury. In the last few years our laboratories extensively investigated the purported neuroprotective role of PACAP in a diffuse model of experimental TBI. First, we discovered that the administration paradigm proved successful in experimental stroke (125 g pre-injury, iv) did not exert beneficial effects in terms of inhibiting traumatically evoked axonal injury; however, intracerebroventricular (icv) injection of the same dose revealed significant axonoprotection [16,31]. We also established the dose-response curve for icv administration of PACAP, demonstrating that 100 g PACAP significantly reduced the density of damaged axons in the corticospinal tract. Further, our most recent observations indicate that a considerable therapeutic window exists for post-injury PACAP-treatment in diffuse experimental TBI, considering that significant axonoprotection could be observed when PACAP was administered 2 h post-injury [46]. On the basis of the above detailed observations PACAP is now considered as a potential candidate for clinical studies in TBI. In a PACAP-deficient mouse model of neuronal injury Armstrong and colleagues found that although motor neuron survival after axotomy was not significantly different in PACAP deficient vs. wild type mice, recovery of axon regeneration after crush injury was significantly delayed. This observation raises the novel hypothesis that endogenous PACAP is critically involved in a carefully controlled immune response that is necessary for proper nerve regeneration after injury [1]. Little is known about the functions of PACAP in humans. The presence of PACAP has been shown in rat and human blood. Radioimmunoassay (RIA) analysis of rat serum has revealed that PACAP-38 concentration is higher in the hypophyseal portal blood than in the peripheral circulation [15]. We have previously identified PACAP-38 in the rat plasma by mass spectrometry and provided evidence that PACAP is released from activated capsaicin-sensitive afferents into the circulation [20,29]. In light of the above the aims of our present study were (1) to determine the presence of PACAP in the cerebrospinal fluid (CSF) and the plasma of patients with severe TBI, (2) to compare PACAP levels in sTBI with controls and to gain an initial insight into the time course of PACAP levels after sTBI and (3) to implement pilot statistical analysis whether there is any significant correlation between the PACAP levels and the clinical outcome.
Patients and methods Study population characteristics Human blood and CSF samples were taken daily from 38 patients who suffered severe TBI and were admitted to Department of Neurosurgery, University of Pecs, Hungary. The main inclusion criterion was Glasgow Coma Scale (GCS) ≤8 on admission caused by sustained severe TBI less than 24 h before the enrollment. Exclusion criteria were age <18, known autoimmune disease, and pregnancy. Because of the comatose state of these patients informed consents were obtained from legally authorized representatives. The patient enrollment, sample collection, and management were carried out with the permission of the local Institutional Review Board (IRB) rigorously complied with the relating rules of the Hungarian law and the Good Clinical Practice.
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Outcome of the patients was defined as survived or not survived of the first week post-injury. Sample collection The first samples were collected at the time of enrollment and then daily until the time of death or for a maximum duration of 10 days. Samples were only obtained by trained individuals using aseptic techniques and extreme care. For taking plasma purple top tubes (K2 EDTA), for collecting CSF red top tubes (plain) were used. Tubes were sat at room temperature for 5–10 min, and the samples were then centrifuged at 4000 rpm for 8 min to gain the plasma and clear CSF. Plasma and CSF were apportioned into 1.8 ml cryovials (aliquot). Fourteen control plasma samples were collected from healthy volunteers with the previously described manner. Our 8 control CSF samples derived from lumbar puncture of patients who were punctured with suspected neurological diseases (e.g.: meningitis) but found negative. PACAP-38 level measurement PACAP-38-like immunoreactivity in the human plasma and CSF was determined with a specific and sensitive RIA technique developed in our laboratory [20,29] and concentrations of the peptide were calculated with a calibration curve. The peptide was extracted from 3 ml plasma samples by addition of a double volume of absolute alcohol and 20 l 96% acetic acid. After precipitation and a second centrifugation (3000 rpm for 20 min at 4 ◦ C) the samples were dried under nitrogen flow and resuspended in 300 l assay buffer to achieve a 10 times higher concentration for the RIA procedure [20,29]. For RIA analysis, 10 l 96% acetic acid was added to 1 ml sample and incubated in 40 ◦ C water bath for 5 min to precipitate the protein content. RIA analysis using the “881113” PACAP-38 antiserum was performed as previously described [20,29]. PACAP-38 concentrations of the samples were read from a calibration curve created using the standard preparations. For statistical purposes we determined the following PACAP-38 levels in the CSF and plasma samples of each patient: first CSF and plasma level, CSF and plasma levels in the second post-traumatic day, highest CSF and plasma levels, average of all CSF and plasma levels. Statistical analysis All measured PACAP levels are in fmol/ml ± SD. To compare plasma and CSF levels of PACAP-38 of the patients with the healthy controls we utilized one way ANOVA with Scheffé post hoc test. In attempt to find correlation between the previously described PACAP-38 levels and the first week mortality we utilized univariate logistic regression analysis. All statistical procedures were carried out by IBM SPSS Statistics 22 for Windows software. Statistical results were considered significant when p < 0.05. Results We enrolled 38 subjects with sustained sTBI into the current study. The age range was between 18 and 83 years with a mean age of 57.97 ± 17.70 years (11 females and 27 males). The on admission GCS was 4.78 ± 1.64. The descriptive statistical analysis of our raw data already showed that all the CSF and plasma PACAP levels were markedly elevated compared to the normal controls although we could observe a highly increased standard deviation at the same time. Table 1 summarizes the results of the descriptive statistical analysis.
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Table 1 Descriptive statistical parameters of PACAP-38 levels. (All PACAP-38 levels are expressed in fmol/ml.).
First CSF Mean CSF Highest CSF Day2 CSF Control CSF First Pl Mean Pl Highest Pl Day2 Pl Control Pl
n
Mean
Median
SD
Minimum
Maximum
26 34 34 25 8 35 38 38 30 14
25.262 28.978 39.677 32.363 16.090 30.263 37.458 51.401 38.788 14.514
24.700 28.965 36.850 32.300 16.067 28.100 36.461 51.000 34.862 14.650
14.134 10.116 19.421 14.220 1.018 13.243 12.463 16.564 18.793 3.080
5.000 8.326 14.000 10.800 14.600 10.700 13.600 19.700 14.400 10.700
79.400 46.645 83.031 83.031 17.450 68.200 68.325 92.800 89.500 21.000
Table 2 Results of univariate logistic regression to the first week mortality. Significant connections revealed with the mean plasma and day2 plasma concentrations. (OR = odds ratio; CI = confidence interval; R2 = Nagelkerke R2 levels.).
First CSF Mean CSF Highest CSF Day 2 CSF First plasma Mean plasma Highest plasma Day 2 plasma
OR (95% CI)
p
R2
1.028 (0.970–1.090) 0.980 (0.905–1.061) 0.999 (0.958–1.041) 1.015 (0.952–1.084) 1.032 (0.977–1.090) 1.143 (1.032–1.267) 1.039 (0.991–1.088) 1.057 (1.003–1.114)
0.348 0.614 0.946 0.643 0.262 0.010 0.110 0.037
0.040 0.011 0.000 0.013 0.048 0.403 0.102 0.256
Discussion For the comparison of sTBI and control PACAP levels in CSF and Pl we utilized one way ANOVA and found significant differences p = 8.45 × 10−13 in case of Pl and p = 6.33 × 10−5 in case of CSF. The Scheffé post hoc test revealed significant difference between control CSF and highest CSF levels and also significant differences were proven between control Pl levels and the first, mean, highest and day 2 plasma levels (Fig. 1). Univariate logistic regression analysis revealed significant connection between the mean and the second post-injury day plasma PACAP levels and the first week mortality (Table 2). To gain further insight into the connection between PACAP levels and the outcome we investigated the relationship between the time course of CSF and plasma PACAP levels and the outcome (Fig. 2). Although we were not able to gain statistically significant difference a clear tendency was well observable that the average plasma levels of the subjects who died within the first post injury week were almost doubled during the entire time period compared to the CSF levels of the same subjects and the CSF and plasma levels of those who survived.
Fig. 1. CSF (A) and Plasma (B) levels of PACAP compared to the controls. Asterisks show the revealed significant difference between highest CSF levels and the controls. In case of plasma concentrations all sTBI levels showed significant difference from the controls. (All measured levels are in fmol/ml.)
In our present study we confirmed that PACAP existed both in plasma and CSF of severe TBI patients as well as non-head injured human controls. Elevated plasma and CSF levels were found in case of severe head injury compared to the controls in each biofluid. Although we were able to find significant connection between plasma PACAP levels and the first week mortality we failed to establish any relationship with clinical parameters. As far as the time course of PACAP levels are concerned, we detected a clear tendency to have a higher plasma PACAP level in case of subjects who died within the first week. Although our results raise several different questions, probably the most important one is the source of the extra amount of PACAP in the biofluids of the sTBI patients compared to the normal controls during the entire investigational period. There are many different pathomechanisms – or a combination of them – which may play a part in the background of this finding.
Fig. 2. Time courses of PACAP38 plasma and CSF levels of all the subjects during the ten days post-injury period (A) and time courses of the plasma and CSF levels of PACAP38 during the first five days post-injury—the lethal cases during the first week compared to the survivors (B). (All measured levels are in fmol/ml.)
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From a simple mechanistical point of view it may seem elegant to account the elevated PACAP levels in the sTBI patients’ biofluids to the primary tissue loss at the time point of the injury. This theory is also supported by the fact that significant decrease of PACAP38 immunoreactivity was found in the primary lesion site in human pathologic samples [47]. However, considering the fact that the estimated half-life of PACAP38 in the human blood is not more than 5–10 min [24,48], only secondary injuries can be responsible for the observed prolonged elevation. As a number of different studies have confirmed the neuroprotective effect of PACAP [2,30,31,33,38,42,43], it seemed reasonable to explain the elevated PACAP levels of sTBI subjects with an endogenous overproduction of PACAP as a pathophysiological response to the loss of neural tissue in the CNS. Landeghem et al. described the cellular localization of PACAP in human brain after TBI. They found a prolonged decrease in PACAP38 content in the lesion site, while a significant increase of PACAP38 positive cells was detected in the penumbra region [47]. A substantially weak point of this concept is that it does not efficiently explain the markedly high concentrations of PACAP in the plasma of the subjects – especially in the lethal cases – during the first post-injury week. In the background of the elevated plasma PACAP levels the damage of the blood–brain barrier (BBB) may be suspected, which – as it is widely described – is very commonly associated with sTBI [9,36,40]. Moreover, the extent of BBB disruption can be quantified based upon the quotient of CSF and serum albumin concentration and biomarker molecules like S100 protein [6]. This logic concept has at least two limitations: (1) although it probably plays a significant role in the increase of PACAP in the peripheral blood, it simply physically cannot be responsible for the even higher PACAP concentrations in the plasma then in CSF—as this phenomenon is clearly visible in Fig. 2B. (2) While PACAP38 has its own long described transport mechanisms even through the intact BBB [4], results from our lab previously showed that its intravenous administration was ineffective in contrast to an icv administration—in an experimental setting of sTBI [16]. As systemic inflammatory response is a quite common complication associated with sTBI [10], frequently even leading to systemic inflammatory response syndrome (SIRS) [25]. PACAP has a well described anti-inflammatory potential [12,13,26]—the elevated PACAP38 levels in sTBI patients biofluids can be explained by an endogenous response to the exaggerated immune reaction. Furthermore, this theory supports the tendency of markedly higher plasma levels in the lethal cases probably due to a more severe systemic inflammatory response than in the survivors. An interesting side stream of the considerations regarding the possible pathomechanisms behind the elevated plasma PACAP38 levels is the potential role of ceruloplasmin. Ceruloplasmin was identified as the binding factor of PACAP38 in the human plasma [44] and the level of ceruloplasmin was also associated with TBI as a potential biomarker [11]. Relatively low levels of ceruloplasmin were described as a predictor of intracranial pressure elevations—it cannot be ruled out that the low ceruloplasmin levels controversially lead to more elevated plasma PACAP38 concentrations in the more severe (lethal) cases. The fact that a significant connection was found between plasma PACAP38 levels and the first week mortality supports PACAP’s potential role as a biomarker. However, it should be emphasized that as only the plasma concentration of the second post-injury day and the average plasma values were significantly predictive, PACAP appears much less effective than the current best set of protein biomarker candidates of TBI like GFAP or UCH-L1 [14]. Nevertheless, our results still draw the attention to the necessity of further more precisely focused investigations regarding PACAP38 as a potential biomarker in large cohorts of subjects. First of
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all – compared to the above described short half-life of PACAP with our hereby utilized sampling protocol – the applicability of PACAP38 as an acute biomarker of the primary tissue loss seems worth to be tested by studies which involve blood sampling at the injury scene (by the ambulance). Further clarification of the role and/or cause of the elevated blood and CSF PACAP38 levels in the pathophysiology of sTBI may reveal that PACAP could be a biomarker of a subprocess like extent of the BBB disruption, initiation of endogenous neuroprotective processes or the state of the systemic inflammatory response. We must draw the attention to substantial limiting factors that may affect our presented results. The number of patients as well as the number of centers could be increased along with a more frequent sampling. This protocol would increase the data available to ensure a more detailed insight into the time course of a molecule with rapid turnover in body fluids. In spite of its shortcomings, we believe that our study contributes to a better understanding of the possible role(s) of PACAP in human sTBI and could serve as a good source for multi-center clinical trials which involve this topic. Conclusions To the best of our knowledge this is the first study where the presence of PACAP and its level in the plasma and in the CSF was measured in TBI patients; our results represent the first publication of the finding that PACAP should be detectable both in the plasma and in the CSF of the severely head injured. Both the plasma and the highest CSF levels showed significant increase compared to the controls. Although our results considered preliminary and limited by many factors (relatively small number of cases, single site) we were able to prove significant connection between plasma PACAP levels and the first week mortality. In our opinion the hereby presented data underlines the necessity of further investigations regarding the potential beneficial or deleterious effects of increased PACAP CSF and blood levels after severe head injury in the near future. Conflicts of interest statement The authors hereby declare that no competing financial interests exist. Acknowledgements This study was supported by National Science Research Found OTKA PD72240, OTKA K104984, the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of SROP 4.2.4.A/2-11-1-2012-0001, SROP-4.2.2.A-11/1/ KONV-2012-0017, SROP-4.2.2.A-11/1/KONV-2012-0024; ‘National Excellence Program’; Magyary Zoltan Scholarship; Arimura Foundation; MTA-PTE “Lendület” Program and Hungarian Brain Research Program—Grant No. KTIA 13 NAP-A-III/5 as well as Hungarian Brain Research Program—Grant No. KTIA 13 NAP-A-II/8. References [1] Armstrong B, Abad C, Chhith S, Cheung-Lau G, Hajji O, Nobuta H, et al. Impaired nerve regeneration and enhanced neuroinflammatory response in mice lacking pituitary adenylyl cyclase activating peptide. Neuroscience 2008;151:63–73. [2] Atlasz T, Babai N, Kiss P, Reglodi D, Tamás A, Szabadfi K, et al. Pituitary adenylate cyclase activating polypeptide is protective in bilateral carotid occlusioninduced retinal lesion in rats. Gen Comp Endocrinol 2007;153:108–14. [3] Babai N, Atlasz T, Tamás A, Reglödi D, Tóth G, Kiss P, et al. Degree of damage compensation by various PACAP treatments in monosodium glutamate-induced retinal degeneration. Neurotox Res 2005;8:227–33. [4] Banks WA, Kastin AJ, Komaki G, Arimura A. Passage of pituitary adenylate cyclase activating polypeptide1-27 and pituitary adenylate cyclase activating polypeptide1-38 across the blood–brain barrier. J Pharmacol Exp Ther 1993;267:690–6.
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[5] Beal M, Palomo T, Kostrzewa R, Archer T. Neuroprotective and neurorestorative strategies for neuronal injury. Neurotox Res 2000;2:71–84. [6] Blyth BJ, Farhavar A, Gee C, Hawthorn B, He H, Nayak A, et al. Validation of serum markers for blood–brain barrier disruption in traumatic brain injury. J Neurotrauma 2009;26:1497–507. [7] Bramlett H, Dietrich W. Pathophysiology of cerebral ischemia and brain trauma: similarities and differences. J Cereb Blood Flow Metab 2004;24:133–50. [8] Chen W, Tzeng S. Pituitary adenylate cyclase-activating polypeptide prevents cell death in the spinal cord with traumatic injury. Neurosci Lett 2005;384:117–21. [9] Chodobski A, Zink BJ, Szmydynger-Chodobska J. Blood–brain barrier pathophysiology in traumatic brain injury. Transl Stroke Res 2011;2:492–516. [10] Das M, Mohapatra S, Mohapatra SS. New perspectives on central and peripheral immune responses to acute traumatic brain injury. J Neuroinflammation 2012;9:12. [11] Dash PK, Redell JB, Hergenroeder G, Zhao J, Clifton GL, Moore A. Serum ceruloplasmin and copper are early biomarkers for traumatic brain injury-associated elevated intracranial pressure. J Neurosci Res 2010;88:1719–26. [12] Delgado M, Abad C, Martinez C, Juarranz MG, Leceta J, Ganea D, et al. PACAP in immunity and inflammation. Ann N Y Acad Sci, 992; 2003. p. 141–57. [13] Delgado M, Martinez C, Pozo D, Calvo JR, Leceta J, Ganea D, et al. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activation polypeptide (PACAP) protect mice from lethal endotoxemia through the inhibition of TNFalpha and IL-6. J Immunol 1999;162:1200–5. [14] Diaz-Arrastia R, Wang KK, Papa L, Sorani MD, Yue JK, Puccio AM, et al. Acute biomarkers of traumatic brain injury: relationship between plasma levels of ubiquitin C-terminal hydrolase-L1 and glial fibrillary acidic protein. J Neurotrauma 2014;31:19–25. [15] Dow R, Bennie J, Fink G. Pituitary adenylate cyclase-activating peptide-38 (PACAP)-38 is released into hypophysial portal blood in the normal male and female rat. J Endocrinol 1994;142:R1–4. [16] Farkas O, Tamas A, Zsombok A, Reglodi D, Pal J, Buki A, et al. Effects of pituitary adenylate cyclase activating polypeptide in a rat model of traumatic brain injury. Regul Pept 2004;123:69–75. [17] Feng S, Kong X, Guo S, Wang P, Li L, Zhong J, et al. Treatment of spinal cord injury with co-grafts of genetically modified Schwann cells and fetal spinal cord cell suspension in the rat. Neurotox Res 2005;7:169–77. [18] Garcia de Yebenes J, Yebenes J, Mena M. Neurotrophic factors in neurodegenerative disorders: model of Parkinson’s disease. Neurotox Res 2000;2:115–37. [19] Gerlach M, Double K, Youdim M, Riederer P. Strategies for the protection of dopaminergic neurons against neurotoxicity. Neurotox Res 2000;2:99–114. [20] Helyes Z, Pozsgai G, Börzsei R, Németh J, Bagoly T, Márk L, et al. Inhibitory effect of PACAP-38 on acute neurogenic and non-neurogenic inflammatory processes in the rat. Peptides 2007;28:1847–55. [21] Katahira M, Yone K, Arishima Y, Nagamine T, Komiya S, Iwata S, et al. The neuroprotective effects of PACAP on spinal cord injury (SCI) in rats. Regul Pept 2003;115:49. [22] Kim W, Kan Y, Ganea D, Hart R, Gozes I, Jonakait G. Vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide inhibit tumor necrosis factor-alpha production in injured spinal cord and in activated microglia via a cAMP-dependent pathway. J Neurosci 2000;20:3622–30. [23] Langlois J, Rutland-Brown W, Thomas K. Traumatic brain injury in the United States: emergency department visits, hospitalizations, and deaths. Atlanta, GA: Centers for Disease Control and Prevention, National Center for Injury Prevention and Control; 2006. [24] Li M, Maderdrut JL, Lertora JJL, Batuman V. Intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP) in a patient with multiple myeloma and myeloma kidney: a case study. Peptides 2007;28:1891–5. [25] Lu J, Goh SJ, Tng PYL, Deng YY, Ling EA, Moochhala S. Systemic inflammatory response following acute traumatic brain injury. Front Biosci, Landmark 2009;14:3795–813. [26] Martinez C, Arranz A, Juarranz Y, Abad C, Garcia-Gomez M, Rosignoli F, et al. PAC1 receptor: emerging target for septic shock therapy. Ann N Y Acad Sci, 1070; 2006. p. 405–10. [27] Miyata A, Arimura A, Dahl R, Minamino N, Uehara A, Jiang L, et al. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 1989;164:567–74.
[28] Murray CJL, Lopez AD. Alternative projections of mortality and disability by cause 1990–2020: global burden of disease study. Lancet 1997;349:1498–504. [29] Németh J, Reglödi D, Pozsgai G, Szabó A, Elekes K, Pintér E, et al. Effect of pituitary adenylate cyclase activating polypeptide-38 on sensory neuropeptide release and neurogenic inflammation in rats and mice. Neuroscience 2006;143:223–30. [30] Racz B, Reglodi D, Kiss P, Babai N, Atlasz T, Gabriel R, et al. In vivo neuroprotection by PACAP in excitotoxic retinal injury: review of effects on retinal morphology and apoptotic signal transduction. Int J Neuroprot Neurodeg 2006;2:80–5. [31] Reglodi D, Tamas A, Lubics A, Szalontay L, Zsombok A, Farkas O, et al. Recent results on the neuroprotective effects of PACAP in rat models of focal cerebral ischemia, Parkinson’s disease and traumatic brain injury. Neurotox Res 2005;8:317. [32] Reglodi D, Tamás A, Lubics A, Szalontay L, Lengvári I. Morphological and functional effects of PACAP in 6-hydroxydopamine-induced lesion of the substantia nigra in rats. Regul Pept 2004;123:85–94. [33] Reglodi D, Tamás A, Somogyvári-Vigh A, Szántó Z, Kertes E, Lénárd L, et al. Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia. Peptides 2002;23: 2227–34. [34] Reglödi D, Lubics A, Kiss P, Lengvári I, Gaszner B, Tóth G, et al. Effect of PACAP in 6-OHDA-induced injury of the substantia nigra in intact young and ovariectomized female rats. Neuropeptides 2006;40:265–74. [35] Rácz B, Gallyas FJ, Kiss P, Tóth G, Hegyi O, Gasz B, et al. The neuroprotective effects of PACAP in monosodium glutamate-induced retinal lesion involve inhibition of proapoptotic signaling pathways. Regul Pept 2006;137: 20–6. [36] Saw MM, Chamberlain J, Barr M, Morgan MP, Burnett JR, Ho KM. Differential disruption of blood–brain barrier in severe traumatic brain injury. Neurocrit Care 2014;20:209–16. [37] Segura-Aguilar J, Kostrzewa R. Neurotoxins and neurotoxicity mechanisms. An overview. Neurotox Res 2006;10:263–87. [38] Shioda S, Ohtaki H, Nakamachi T, Dohi K, Watanabe J, Nakajo S, et al. Pleiotropic functions of PACAP in the CNS: neuroprotection and neurodevelopment. Ann NY Acad Sci 2006;1070:550–60. [39] Shioda S, Ozawa H, Dohi K, Mizushima H, Matsumoto K, Nakajo S, et al. PACAP protects hippocampal neurons against apoptosis: involvement of JNK/SAPK signaling pathway. Ann NY Acad Sci 1998;865:111–7. [40] Shlosberg D, Benifla M, Kaufer D, Friedman A. Blood–brain barrier breakdown as a therapeutic target in traumatic brain injury. Nat Rev Neurol 2010;6:393–403. [41] Skoglösa Y, Lewén A, Takei N, Hillered L, Lindholm D. Regulation of pituitary adenylate cyclase activating polypeptide and its receptor type 1 after traumatic brain injury: comparison with brain-derived neurotrophic factor and the induction of neuronal cell death. Neuroscience 1999;90:235–47. [42] Somogyvári-Vigh A, Reglodi D. Pituitary adenylate cyclase activating polypeptide: a potential neuroprotective peptide. Curr Pharm Des 2004;10:2861–89. [43] Tamas A, Reglodi D, Farkas O, Kovesdi E, Pal J, Povlishock JT, et al. Effect of PACAP in central and peripheral nerve injuries. Int J Mol Sci 2012;13:8430–48. [44] Tams JW, Johnsen AH, Fahrenkrug J. Identification of pituitary adenylate cyclase-activating polypeptide 1-38-binding factor in human plasma, as ceruloplasmin. Biochem J 1999;341:271–6. [45] Tamás A, Lubics A, Lengvári I, Reglódi D. Protective effects of PACAP in excitotoxic striatal lesion. Ann NY Acad Sci 2006;1070:570–4. [46] Tamás A, Zsombok A, Farkas O, Reglödi D, Pál J, Büki A, et al. Postinjury administration of pituitary adenylate cyclase activating polypeptide (PACAP) attenuates traumatically induced axonal injury in rats. J Neurotrauma 2006;23:686–95. [47] van Landeghem FKH, Weiss T, Oehmichen M, von Deimling A. Cellular localization of pituitary adenylate cyclase-activating peptide (PACAP) following traumatic brain injury in humans. Acta Neuropathol 2007;113:683–93. [48] Vaudry D, Falluel-Morel A, Bourgault S, Basille M, Burel D, Wurtz O, et al. Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery. Pharmacol Rev 2009;61:283–357. [49] Vaudry D, Gonzalez B, Basille M, Yon L, Fournier A, Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 2000;52:269–324.