Eurasian J Med 2019; 51(1): 42-8 Original Article

Effects of versus on Post-Ischemic Brain Injury and Edema

Betul Can1 , Semih Oz2 , Varol Sahinturk3 , Ahmet Musmul4 , İbrahim Ozkan Alatas1

ABSTRACT Objective: The aim of this study was to compare the effects of conivaptan, an arginine antago- nist, and mannitol, a sugar alcohol, on cerebral -induced brain injury and edema in rats. Materials and Methods: Fifty-eight 8-week-old male Sprague Dawley rats were randomly divided into five groups: control, ischemia–reperfusion (I/R)+, I/R+mannitol, I/R+10 mg/ml conivaptan, and I/R+20 mg/ ml conivaptan. Cerebral ischemia was induced by common carotid artery occlusion for 30 minutes. Saline, mannitol, or conivaptan were administered intravenously at the onset of reperfusion. Blood and brain tissue samples were taken at the 6th hour of reperfusion. The electrolytes (Na+–K+–Cl−), osmolality, arginine va- sopressin, albumin, progranulin (PGRN), neuron-specific enolase (NSE), and myeloperoxidase activity were measured in rat serum samples. Brain frontal/hippocampal sections were stained with hematoxylin–eosin and TUNEL techniques to evaluate histopathological changes. Results: Statistical analyses revealed that conivaptan caused significant changes in the electrolyte, NSE, and PGRN levels and osmolality when compared with mannitol. Conivaptan treatment showed positive effects on serum biochemistry and tissue histology. Cite this article as: Can B, Oz S, Sahinturk V, Musmul A, Alatas IO. Effects of Conivaptan versus Conclusion: Our findings revealed that conivaptan shows more diuretic activity than mannitol and triggers Mannitol on Post-Ischemic Brain Injury and Edema. neither any damages nor edema in the brain tissue. This study may provide beneficial information for the Eurasian J Med 2019; 51(1): 42-8. development of treatment strategies for ischemia-related cerebrovascular diseases. ORCID IDs of the authors: Keywords: Brain injury, conivaptan, ischemia–reperfusion, mannitol, neuronal damage B.C. 0000-0001-8430-5298 S.O. 0000-0001-1543-2223 V.S. 0000-0003-2317-3644 A.M. 0000-0003-3669-7017 Introduction I.O.A 0000-0002-1753-8873 The formation of brain edema (BE), a continuous process with an intense intracellular and extra-

1Department of Medical Biochemistry, Eskisehir cellular ion and water exchange [1], is one of the most important acute/subacute complications Osmangazi University School of Medicine, of cerebral injuries. Changes in intra- and extracellular volumes may threaten the regional or Eskisehir, Turkey global cerebral blood flow and cell metabolism; given the fixed volume of the skull, this can lead 2Department of Health Services, Eskisehir Osmangazi University Vocational School of to critical consequences due to the compression of vital brain structures [2]. Health Services, Eskisehir, Turkey 3Department of Histology and Embryology, Eskisehir Osmangazi University School of The osmotherapy applied as a part of the medical treatment algorithms is of great importance in Medicine, Eskisehir, Turkey the management of and increased (ICP) following brain injury 4Department of Medical Services and [3]. Mannitol, a widely used osmotic diuretic agent for the treatment of BE and high ICP for many Techniques, Eskisehir Osmangazi University Vocational School of Health Services, Eskisehir, years [4], cannot meet the anticipated efficacy because it can cause serious side effects and more Turkey fluid to be drawn into the tissues [5]. Therefore, the need for further research is strongly empha- sized in the literature to detect an ideal anti-edema agent that has a low number of side effects [2]. Received: October 22, 2018 Accepted: December 20, 2018 Arginine vasopressin (AVP), a neurohypophysial antidiuretic hormone, has many functions, such Correspondence to: Betul Can E-mail: [email protected] as the regulation of free-water reabsorption and body fluid homeostasis, and it acts upon its spe-

cific G protein-coupled receptors defined as V1A (V1, vascular), V2 (renal), and V1B (V3, pituitary) DOI 10.5152/eurasianjmed.2019.18368 [6]. It has been suggested that the AVP hypersecretion plays a critical role in the BE formation, leading to vasospasm, water retention, dilutional , and low plasma osmolality [7]. AVP receptors might be also preferred as a crucial therapeutic target [8]. Content of this journal is licensed under a Creative Commons Vaptans, a new group of drugs that blocks the AVP receptors, were recommended for the Attribution 4.0 International License. treatment of diseases accompanied by water retention [9]. Conivaptan, one of these V1A/V2 Eurasian J Med 2019; 51(1): 42-8 Can et al. The Effects of Conivaptan on Post-Ischemic Brain Injury and Edema • 43 receptor antagonists, was approved by the Food Anesthesia Preparation and analysis of serum samples and Drugs Administration in 2005 for the treat- Prior to surgical procedures, the animals All blood samples were centrifuged at 1000 x ment of patients with clinical hyponatremia [10]. were deprived of food for approximately g for 5 minutes. Serum electrolytes (Na+, K+, Conivaptan has aquaretic effects, and as such, it 12 hours. On the day of surgery, their body Cl−), albumin, and osmolality measurements has a high affinity for the2 V receptors in renal weight was measured. The anesthesia was were conducted immediately on the same day, collecting ducts, promoting renal free-water performed by intramuscular injection of ket- and the remaining serum samples were main- excretion without having a significant effect on amine HCl (50 mg/kg), which is an anesthet- tained at −80ºC until the other biochemical electrolyte excretion [11]. However, the ben- ic/analgesic agent for painful procedures, and analysis. The electrolyte levels in serum samples efits of AVP receptor antagonists on the brain xylazine HCl (10 mg/kg), which is a muscle were measured using the Auto Analyzer Sys- edema process, when applied at the onset of relaxant. At the end of the reperfusion pro- tem (Roche Cobas ISE modular; ISE-indirect) reperfusion, have not been clarified. cess, the animals were anesthetized intra- and expressed as mmol/L. Serum albumin was peritoneally again with the same agents and measured using the Roche Cobas C modular This study, designed with the purpose of obtain- doses, and blood and brain tissue specimens and was expressed as g/dL. Serum osmolality ing the above-mentioned scientific data, was were taken immediately. was measured using a vapor pressure osmom- aimed to compare the effects of conivaptan and eter (Vapro Model 5600, Wescor Inc., USA) mannitol treatments on the post-ischemic brain Experimental design and expressed as mmol/kg. injury and BE in the acute phase. In the litera- The rats were randomly divided into the fol- ture, to the best of our knowledge, there are no lowing five groups: control (sham; n=10), isch- The serum AVP, neuron-specific enolase (NSE), studies that compare the effects of mannitol and emia–reperfusion (I/R)+saline (I/R; n=12), I/ and progranulin (PGRN) levels were measured conivaptan with this regard. R+mannitol (MAN; n=12), I/R+conivaptan 10 with the help of the commercial ELISA kits, ac- mg/ml (CON10; n=12), and I/R+conivaptan 20 cording to the manufacturer’s instructions, us- Materials and Methods mg/ml (CON20; n=12). One animal in the I/R ing a Plate Reader (VICTOR X3, PerkinElmer All research protocols in this study were ap- group was eliminated from the study due to re- Inc., USA) at an optical density of 450 nm. The proved by the Institutional Ethics Committee spiratory arrest during reperfusion. AVP ELISA kit was based on a competitive inhi- (Protocol Number: 560-1/2018). bition enzyme immunoassay technique for the Our study was based on a two-vessel occlu- quantitative measurement of AVP in rat serum Chemicals and kits sion model with a slight modification [12]. Un- samples. The other two ELISA kits were based The following chemicals were used in the like this model, procedures were on a quantitative sandwich enzyme immunoas- study: conivaptan hydrochloride (cat. #TRC- not performed in our study because of the say technique. The detection range, minimum C384700) purchased from Toronto Research diuretic/aquaretic characteristics of the admin- detectable dose, and intra- and inter-assay Chemicals (Canada), dimethyl sulfoxide (cat. istered agents and the necessity of minimizing precisions of all the ELISA kits were as follows, #D5879), D-mannitol (cat. #M4125), hydro- the surgical manipulation to avoid the risk of respectively: 12.35-1,000 pg/mL, 4.63 pg/mL, gen peroxide (cat. #18312), and 3,3’,5,5’-etra- infection, which may affect the results. In brief, and CV<10% and CV<12% for the AVP kit; methylbenzidine dihydrochloride hydrate (cat. we induced cerebral ischemia by clamping ca- 0.625-40 ng/mL, 0.237 ng/mL, and CV<8% and #861510) were purchased from Sigma-Aldrich rotid arteries bilaterally using a vascular clamp CV<10% for the NSE kit; and 7.8-500 ng/mL, (USA). Ketamine HCl was purchased from Pfiz- (Vascu Stop Bulldog Clamp, Turkey) for 30 1.95 ng/mL, and CV<10% and CV<12% for the er (USA); xylazine HCl from Egevet (Turkey); minutes after a vertical midline neck incision, PGRN Kit. eosin Y (cat. #109844) and hematoxylin (cat. without any added hypotension procedures. #105174) solutions from Merck (Germany); After 30 minutes, the clamps were removed, As a predictor of leukocyte infiltration and in- rat commercial enzyme-linked immunosorbent and reperfusion was initiated. Saline (0.9% flammation, the serum myeloperoxidase (MPO) assay (ELISA) kits from AVP (cat. #CEB139Ra); NaCl), mannitol (20% wt/vol; dissolved in sa- activity was measured according to the previ- and NSE (cat. #SEA537Ra) from Cloud-Clone line; MW=182.17), and conivaptan (10 or 20 ously reported method [14]. The method is Corp. (USA). The progranulin (cat. #CSB- mg/ml=18.5 or 37 mg kg-1; dissolved in 5% fi- based on the measurement of the oxidation of EL009939RA) ELISA kit was purchased from nal DMSO vol/vol; MW=535.04) were applied a synthetic substrate, 3,3’-5,5’-tetramethylben- Cusabio Biotech Comp. (China). The albumin to the relevant groups by the infusion into the zidine (TMB), by MPO-catalyzed reaction. The assay kit (cat. #3183688122) was supplied from jugular vein for 30 minutes (1 ml/h) at the be- reaction was initiated by the addition of hydro- Roche Diagnostics (Mannheim, Germany), and ginning of reperfusion [13]. After the infusion, gen peroxide to the reaction mixture at 37°C. ApopTag Plus peroxidase in situ apoptosis de- the incision area was sutured, and the operated The increase in absorbance during the reaction tection kits (Cat. #S7101) were from Merck animals were maintained in separate cages. For was monitored using a spectrophotometer (UV- (Germany). the control group rats, only neck incision-sutur- 1601; Shimadzu, Tokyo, Japan) at 655 nm for 5 ation was performed without arterial clamping. minutes, and the serum MPO activity in a minute Animals and housing To prevent hypovolemia, 1 mL of saline was in- was calculated as IU/mL. Fifty-eight 8-week-old male Sprague Dawley jected intraperitoneally to the rats, which were rats weighing 250–300 g were purchased from treated with mannitol and conivaptan, immedi- Preparation and analysis of tissue samples KOBAY Laboratories (Ankara, Turkey) and ately after skin suturing. The cardiac blood and The brain tissue samples were used for the housed at room temperature (21±1°C) and brain tissue samples from rats were taken at histological analysis and the detection of brain under the 12/12 h light/dark cycle in a humidity- the sixth hour of reperfusion (6th hour after water content (BWC). Before the histological controlled (45%–50%) environment with a free skin suturing in the control group) under anes- analysis, half of the rats, which were randomly access to standard rodent food pellets and tap thesia, and the samples were prepared for the selected from the groups, were subjected to water ad libitum. analysis as detailed below. transcardial perfusion–fixation to ensure that 44 • Can et al. The Effects of Conivaptan on Post-Ischemic Brain Injury and Edema Eurasian J Med 2019; 51(1): 42-8 the fixative reaches all regions of the tissue, to Statistical Analysis ml conivaptan when compared to the I/R group reduce the mechanical damage, and to evalu- Data were assessed for the normality using (p<0.001). The CON10 group had the highest ate the tissue histology more accurate [15]. For the Shapiro–Wilk test. Comparisons for vari- osmolality (p<0.001). this purpose, the anesthetized rats were per- ables that consisted of independent measure- fused first with 20 mL of saline followed by ap- ments and showed normal distribution were Serum arginine vasopressin levels proximately 100 mL of freshly prepared neutral evaluated with the one-way analysis of variance Because an AVP receptor antagonist was used formaldehyde solution. The brain tissues were (Tukey’s post-hoc analysis) test, and the com- in this study, the serum AVP levels were also removed meticulously, post-fixed in 10% para- parisons for non-normally distributed variables measured to examine the effects of the treat- formaldehyde solution, and kept in a dark and were analyzed by the Kruskal–Wallis test by a ment on the circulating hormone levels. Serum coolenvironment until the analyses. blinded statistician. The data were shown as a AVP levels were higher in the MAN, CON10, sample size (n), the mean±SD, or median, 25th and CON20 groups compared to the control For the histological analysis, tissue sections were and 75th percentile values. The statistical signifi- (p<0.001). The CON10 group had the highest obtained from the frontal cortex and hippo- cance level was set at p<0.05. Statistical analysis AVP level, and it was significantly higher than the campus using a rat brain slicer according to the was performed using the The Statistical Package I/R group (p<0.001). for the Social Sciences (SPSS) version 21.0 (IBM stereotaxic coordinates. Tissue sections were Corp., Armonk, NY, USA). Serum NSE, progranulin, and albumin levels fixed in 10% formaldehyde, exposed to ascend- Our statistical analyses showed that I/R led ing graded ethyl-alcohols, and embedded in Results to an increase in NSE levels compared to the paraffin. The obtained paraffin blocks were cut Results of the serum analyses are summarized in control, whereas conivaptan caused a signifi- to slices 4–5 µm in thickness. These slices were Table 1, and the frontal and hippocampal micro- cant decrease (p<0.001). Serum progranulin stained with the hematoxylin–eosin (HE) tech- graphs are presented in Figures 1 and 2. levels in the I/R and MAN groups was nearly nique to evaluate the histopathological changes similar to the control, but progranulin levels af- and with TUNEL to display apoptotic cells us- Serum electrolyte (Na+, K+, Cl−) levels and os- ter conivaptan treatment increased more than ing a commercial apoptosis detection kit. After molality in mannitol-treated rats, which was statistically the staining, images were captured using a DP70 According to our results, cerebral I/R caused a significant (p<0.01). The serum albumin levels digital camera (Olympus Corp., Tokyo, Japan) significantly decrease in the serum Na+ levels showed no significant difference among the attached to a BX51 light microscope (Olympus when compared with the control (p<0.001). groups (p>0.05). Corp., Tokyo, Japan). Although both mannitol and conivaptan in- creased the Na+ levels, the conivaptan group Serum MPO activity To detect the BWC, fresh brain tissues ob- apparently had higher Na+ levels than the MAN Although the MPO activity was higher in the I/R tained from non-perfused rats (other half of group (p<0.001). There was no significant dif- group than in others, there was no significant dif- the groups) weighed immediately (wet weight), ference in K+ levels among the groups (p>0.05). ference detected among the groups (p>0.05). dried at approximately 100°C for 24 hours us- In addition, serum Cl− levels decreased in the ing an incubator (Binder BD 53, Binder GmbH, I/R group compared to the control significantly BWC Germany) and reweighted (dry weight). The (p<0.001). However, the conivaptan group had The percentage of water content of the whole- percentage of BWC was calculated by the fol- higher Cl− levels than other groups (p<0.001). brain tissues was very close between the groups. lowing formula as previously described [16]: In addition, serum osmolality showed a decline However, a decline was observed in other

H2O%=(1−dry weight/wet weight)×100. after the treatment with mannitol and 20 mg/ groups when compared with control (p<0.01).

Table 1. Summary of Statistical Data for Blood Serum Parameters of the Study Groups CONTROL (n=10) I/R (n=11) MAN (n=12) CON10 (n=12) CON20 (n=12) p Na (mmol/L) 145.60±2.55 141.55±3.56a 142.92±3.90b 149.17±3.38b, c 148.00±4.57b, c <0.001* K (mmol/L) 6.40 (6.04-7.45) 6.46 (5.87-6.69) 6.42 (6.04-6.94) 6.09 (5.93-6.29) 6.82 (6.25-7.54) ns** Cl (mmol/L) 103.31±2.40 101.17±1.81a 100.13±4.01 107.93±3.75a, b, c 106.60±4.72a, b, c <0.001* Albumin (mg/dL) 3.72±0.18 3.58±0.28 3.63±0.25 3.76±0.27 3.84±0.31 ns* Osmolality (mmol/kg) 345.55±20.58 360.00±14.62 321.08±12.85b 376.13±41.61c 319.17±18.74b, c, d <0.001* AVP (pg/mL) 28.07±5.73 31.53±5.81 35.82±6.82a 39.67±8.76a, b 36.40±4.65a <0.001* Progranulin (ng/mL) 13.74 (10.41-19.26) 13.95 (12.47-18.25) 12.13 (10.70-13.21) 15.87 (13.90-18.90) c 17.08 (14.22-22.41) c <0.01** NSE (ng/mL) 3.96±0.80 4.39±0.68 4.45±0.80 3.25±0.67b, c 3.41±0.86b, c <0.001* MPO activity (U/mL) 0.76±0.30 0.82±0.66 0.56±0.39 0.46±0.28 0.72±0.68 ns*

a a a Brain water content (H2O%) 78.47±0.29 77.93±0.39a 77.71±0.47 77.61±0.42 77.47±0.21 <0.01* * One-way analysis of variance (data are expressed as the mean±standard deviation)

** Kruskal–Wallis one-way analysis of variance (data are expressed as the median, 25th and 75th percentiles)

n, sample size; ns, not significant; AVP, arginine vasopressin; NSE, neuron-specific enolase; MPO, myeloperoxidase

a,b,c,d Significant difference versus the control, I/R, MAN, and CON10 group, respectively. Eurasian J Med 2019; 51(1): 42-8 Can et al. The Effects of Conivaptan on Post-Ischemic Brain Injury and Edema • 45

Hematoxylin–Eosin staining of the frontal cor- a b tex and hippocampus The brain frontal cortex sections stained with HE had a normal histology finding in the control group (Figure 1). In the I/R group, some neu- ron degenerations were observed, whereas in the MAN and CON20 groups, the number of degenerative neurons was lower than in the I/R group. The CON20 group had a nearly normal histology in the frontal sections, and neuron de- generations were minimal than in other treat- ment groups. In the brain hippocampal sections c d stained with HE, the tissue histology was normal, and no edematous areas, neuron degenerations, or inflammatory cells were detected. There was no significant difference with regard to the hip- pocampal histology between the groups.

TUNEL staining of the frontal cortex and hip- pocampus In the TUNEL staining of the brain frontal cor- tex sections, no apoptotic neurons were de- tected in the control group (Figure 2). In the I/R group, many apoptotic neurons were observed, e f and their number was lower in the MAN group compared to the I/R. The CON10 group had a normal histology, and no apoptotic cells in the frontal sections were observed. The CON20 group had fewer degenerated neurons with acidophilic cytoplasm with no apoptosis. In ad- dition, the tissue histology of the brain hippo- campus was almost normal, and there was no difference between the groups (not shown).

Discussion g h In contrast to mannitol, conivaptan caused an increase in the Na+ and Cl− levels In this study, in which electrolyte levels were ex- amined to assess the fluid–electrolyte balance, it was observed that the serum Na+ and Cl− levels in the I/R group decreased significantly com- pared to the control. This may be because the decrease in the Na+–K+–ATPase activity in the + i j I/R process leads to the Na uptake into the cell and also the Cl− uptake to provide an osmotic balance, as described in previous studies [17].

A significant increase in the serum Na+ and Cl− levels in the CON10 and CON20 group suggests that electrolytes may have had vascu- lar involvement, increasing the renal excretion of free water due to the aquaretic nature of Figure 1. a-j. Micrographs taken from the frontal cortex and hippocampal sections of the rat brains, conivaptan. This approach was supported by the stained with hematoxylin–eosin (HE). (a) The control group showed normal histological appearance. + − There were many degenerated neurons with acidophilic cytoplasm in the IR group (b, arrows) and following findings: the Na and Cl levels were a few of degenerated neurons with acidophilic cytoplasm in the MAN group (c, arrow). (d) Normal high, and the brain histology was relatively close histological appearance in the CON10 group. A very few degenerated neurons with acidophilic to the control in the CON10 group. These find- cytoplasm in the CON20 group (e, arrows). In the HE staining of the brain hippocampus sections (f-j), ings suggest that conivaptan, depending on the all of the experimental groups were observed to have a normal tissue histology and no edematous areas, inflammatory cell migrations, or neuronal degenerations. Scale bars for the frontal micrographs dosage, may be more effective in the I/R pro- are 50 µm and for hippocampal 100 µm. cess than mannitol under the same application 46 • Can et al. The Effects of Conivaptan on Post-Ischemic Brain Injury and Edema Eurasian J Med 2019; 51(1): 42-8

time period is needed for an apparent edema a b formation. Interestingly, a decrease in BWC in the I/R group was remarkable. It could be due to the minimal diuretic effect of saline, since the infusion of saline was equal to that of the other groups in the I/R group. In histological examina- tions, neuron degenerations and a large num- ber of neurons presenting apoptosis (TUNEL- positive cells) were observed in the I/R group. In the frontal cortex of the CON10 group, the brain histology was found to be close to the con- trol in general. This demonstrated that 10 mg/ c d mL conivaptan might be efficient in preventing brain tissue damage, and also the positive effects of mannitol treatment in cerebral injury were clearly more limited.

Conivaptan increased AVP levels due to its re- ceptor antagonism It has been reported that AVP plays an impor- tant role in various studies on brain injury, such as cerebral ischemia [18, 24] and trauma [25]. It has been shown that AVP plays an important

role in increasing the vascular tone through V1A e receptors and in the free-water uptake via V2 receptors [11]. In our study, AVP levels in the Figure 2. a-e. Micrographs taken from the MAN and the conivaptan groups were signifi- frontal lobe sections of the rat brains, stained with TUNEL to display apoptotic cells. (a) The cantly increased compared to control. This in- control group showed a normal histological crease in the MAN group occurred probably appearance with no apoptosis. There were many due to the stimulation of water uptake via the apoptotic neurons in the IR group (b, arrows) and a few apoptotic neurons in the MAN mannitol diuresis effect. The reason for the high group (c, arrows). (d, e) Normal histological AVP levels observed in conivaptan groups could appearance with no apoptosis both in the be due to the AVP receptor antagonism, which CON10 and CON20 groups, respectively. Scale means that AVP was not able to bind to its re- bars for the frontal micrographs were 50 µm. ceptors, the water excretion continued due to times and conditions. In addition, there was no shown to play an important role as an antioxi- the aquaretic effects of conivaptan, and thus the significant difference in the+ K levels between dant by antagonizing endogenous and exoge- AVP release was stimulated much more. the groups in accordance with the literature nous oxidative stress sources in the plasma [21]. [18]. In this case, astrocytic end-feet in BBB may In our study, albumin levels were not different Conivaptan decreased the serum NSE levels play a homeostatic role in keeping the K+ levels between the study groups. This may be due to In clinical trials, it was emphasized that the relatively in balance, but no further analysis was the fact that albumin can be damaged during the majority of circulatory biomarkers, which re- conducted in our study to prove this. antioxidant activity but can be rapidly renewed flect post-injury dysfunction in the brain tis- after being proteolytically disrupt [21]. Consid- sue, have an independent diagnostic and prog- Conivaptan serum osmolality change was ering the rapid regeneration of albumin, its lev- nostic significance in ischemic conditions [26]. dose dependent els should be followed in short time-points. One of these important clinical biomarkers is Our results showed that mannitol caused a de- NSE (2-Phospho-D-Glycerat-Hydrolase; EC crease in osmolality compared to the I/R group Conivaptan caused significant changes in 4.2.1.11). In another study, it was reported that possibly due to the electrolyte excretion via its BWC, but they were neither negative nor MCAO-induced cortical infarction and neuro- diuretic effect, whereas the CON10 group had positive logical dysfunction were associated with neuro- a high osmolality and the CON20 group had a In an early stage of edema, the water influx is ex- nal depletion and vascular distribution of NSE low. Changes in the concentrations of Na+ and plained based on the increase in osmotic activ- in the brain, causing an increase in NSE in the Cl− in the CON10 group might have contrib- ity and hydrostatic pressure gradients [22]. In a plasma [27]. In this study, mannitol treatment did uted to an increase in osmolality. study of cerebral edema performed with middle not result in a significant difference in NSE levels cerebral artery occlusion (MCAO) [23], it was compared to the I/R group, consistent with the Albumin levels showed no significant changes found that swelling began to develop 3 hours studies that serum NSE levels did not change among the groups from the occlusion; the increase rapidly peaked after mannitol infusion. Conivaptan treatments Albumin plays a role in limiting the fluid leakage on days 2 and 3, and then began to gradually caused a decrease in NSE levels compared to into the tissue from the vascular system [19], slow down. In our study, BWC did not show the I/R and MAN groups, bringing tissue histol- preserving the plasma colloid oncotic pressure, a significant difference between the groups at ogy close to the control, and they did not have and inducing hemodilution [20]. It has also been the 6th hour of reperfusion. Possibly, a longer a negative effect on tissue damage. Therefore, Eurasian J Med 2019; 51(1): 42-8 Can et al. The Effects of Conivaptan on Post-Ischemic Brain Injury and Edema • 47 it may be considered that conivaptan causes all diseases, but further experimental studies are 11. Wada K, Tahara A, Arai Y, et al. Effect of these changes due to its free-water excretion required. This study can also contribute to the the vasopressin receptor antagonist conivaptan and prevents neuronal NSE losses more than literature providing useful information to design in rats with heart failure following myocardial mannitol. new comprehensive studies. infarction. Eur J Pharmacol 2002; 450: 169-77. [CrossRef] 12. Smith ML, Bendek G, Dahlgren N, Rosen I, Conivaptan increased PGRN levels, contrary Ethics Committee Approval: Ethics Committee ap- Wieloch T, et al. Models for studying long-term to mannitol proval was received for this study from the Ethics recovery following forebrain ischemia in the In a comprehensive study, it was found that Committee of Eskişehir Osmangazi University (560- 1/2018). rat. 2. A 2-vessel occlusion model. Acta Neurol PGRN had a protective effect against ischemia Scand 1984; 69: 385-401. [CrossRef] with various mechanisms such as the mitigation Peer-review: Externally peer-reviewed. 13. Taya K, Gulsen S, Okuno K, Prieto R, Marmarou of BBB destruction, suppression of neuroinflam- CR, Marmarou A. Modulation of AQP4 expres- mation, and neuroprotection [28]. But there is Author contributions: Concept – B.C., Ö.A.; Design sion by the selective V1a receptor antago- no study about the relationship of PGRN levels - B.C., Ö.A.; Supervision - Ö.A.; Resource - B.C., Ö.A.; nist, SR49059, decreases trauma-induced brain with mannitol or with those of the vaptans. In Materials - B.C., Ö.A.; Data Collection and/or Process- edema. Acta Neurochir Suppl 2008; 102: 425-9. our findings, there was a significant increase in ing - B.C.; Analysis and /or Interpretation - B.C., Ö.A., [CrossRef] progranulin levels in the conivaptan groups com- S.Ö., V.Ş., A.M.; Literature Search - B.C.; Writing - B.C.; 14. Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura pared to the MAN group. Conivaptan could be Critical Reviews - B.C., Ö.A. T. Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Anal Biochem more beneficial against the neuronal damage 1983; 132: 345-52. [CrossRef] than mannitol; however, the high standard de- Conflict of Interest:The authors have no conflict of interest to declare. 15. Gage GJ, Kipke DR, Shain W. Whole animal viations observed in the groups make difficult perfusion fixation for rodents. J Vis Exp 2012; to establish whether conivaptan could really be Financial Disclosure: This study was supported by Sci- 65: pii: 3564. [CrossRef] neuroprotective. entific Research Projects Commission of the Eskise- 16. Lin TN, He YY, Wu G, Khan M, Hsu CY. Effect of hir Osmangazi University (Date: 02.06.2017, Project brain edema on infarct volume in a focal cerebral MPO activities did not change after conivap- Number: 1524/2017). ischemia model in rats. 1993; 24: 117-21. tan treatment [CrossRef] Myeloperoxidase, which is an important enzyme References 17. Suhail M. Na(+), K(+)-ATPase: Ubiquitous classified as hydrogen peroxide oxidoreductase 1. Jia SW, Liu XY, Wang SC, Wang YF. Vasopressin Multifunctional Transmembrane Protein and (EC 1.11.1.7), is secreted as the inflammatory Hypersecretion-Associated Brain Edema its Relevance to Various Pathophysiological response to tissue damage and is widely distrib- Formation in Ischemic Stroke: Underlying Conditions. J Clin Med Res 2010; 2: 1-17. [CrossRef] uted in ischemic tissues [29]. In our study, there Mechanisms. J Stroke and Cerebrovasc Dis 2016; 18. Molnar AH, Varga C, Berko A, et al. Inhibitory was no significant difference between the groups 25: 1289-300. [CrossRef] effect of vasopressin receptor antagonist OPC- in MPO activities, and no histological findings 2. Walcott BP, Kahle KT, Simard JM. Novel treatment targets for cerebral edema. Neurotherapeutics 31260 on experimental brain oedema induced proved that the infiltration of the immune cells 2012; 9: 65-72. [CrossRef] by global cerebral ischaemia. Acta Neurochir occurred in the tissue, which was not in compli- 3. Bhardwaj A. Osmotherapy in neurocritical care. (Wien) 2008; 150: 265-71. [CrossRef] ance with the previous study. The migration of Curr Neurol Neurosci Rep 2007; 7: 513-21. 19. Sutherland BA, Minnerup J, Balami JS, Arba F, the inflammatory cells probably did not occur [CrossRef] Buchan AM, Kleinschnitz C. Neuroprotection in the determined time-point in this study. But 4. Knapp JM. Hyperosmolar therapy in the treat- for ischaemic stroke: translation from the bench in our recent study, we showed that conivaptan ment of severe head injury in children: mannitol to the bedside. Int J Stroke 2012; 7: 407-18. decreased pro-inflammatory cytokine (TNF-α and hypertonic saline. AACN Clin Issues 2005; [CrossRef] and IL-15) levels significantly compared to the 16: 199-211. [CrossRef] 20. Ginsberg MD. Neuroprotection for isch- I/R group [30]. In addition, serum MPO activities 5. Wykes V, Vindlacheruvu R. Intracranial pressure, emic stroke: past, present and future. Neuropharmacology 2008; 55: 363-89. in the control group showed that the model we cerebral blood flow and brain oedema. Surgery [CrossRef] used caused no additional infection. (Oxford) 2015; 33: 355-62. [CrossRef] 6. Palmer BF. Vasopressin receptor antago- 21. Halliwell B. Albumin--an important extracellular nists. Curr Hypertens Rep 2015; 17: 510. antioxidant? Biochem Pharmacol 1988; 37: 569- As a result, an early use of conivaptan in post- [CrossRef] 71. [CrossRef] ischemic conditions did not cause any necrotic/ 7. Barreca T, Gandolfo C, Corsini G, et al. Evaluation 22. Stokum JA, Gerzanich V, Simard JM. Molecular apoptotic effects on the brain tissue or had any of the secretory pattern of plasma arginine vaso- pathophysiology of cerebral edema. J inflammatory triggering effects. Due to its abil- pressin in stroke patients. Cerebrovasc Dis 2001; Cereb Blood Flow Metab 2016; 36: 513-38. ity to cause renal free-water excretion, conivap- 11: 113-8. [CrossRef] [CrossRef] tan did not lead to the serum electrolyte loss, 8. Zhao XY, Wu CF, Yang J, et al. Effect of arginine 23. Gotoh O, Asano T, Koide T, Takakura K. Ischemic contrary to mannitol. The beneficial effect of vasopressin on the cortex edema in the ischemic brain edema following occlusion of the mid- mannitol on the brain tissue damage was more stroke of Mongolian gerbils. Neuropeptides dle cerebral artery in the rat. I: The time limited than conivaptan. 2015; 51: 55-62. [CrossRef] courses of the brain water, sodium and potas- 9. Peri A. Clinical review: the use of vaptans in clini- sium contents and blood-brain barrier perme- cal endocrinology. J Clin Endocrinol Metab 2013; ability to 125I-albumin. Stroke 1985; 16: 101-9. In the literature, no studies comparing the effects 98: 1321-32. [CrossRef] [CrossRef] of mannitol and conivaptan have been found in 10. Palmer BF, Rock AD, Woodward EJ. Dose com- 24. Ikeda Y, Toda S, Kawamoto T, Teramoto A. this respect. We concluded that conivaptan, an parison of conivaptan (Vaprisol®) in patients Arginine vasopressin release inhibitor RU51599 arginine vasopressin antagonist, might be consid- with euvolemic or hypervolemic hyponatremia- attenuates brain oedema following transient ered as an alternative to mannitol for the treat- efficacy, safety, and pharmacokinetics. Drug Des forebrain ischaemia in rats. Acta Neurochir ment in the early phases of ischemia-related Devel Ther 2016; 10: 339-51. (Wien) 1997; 139: 1166-71. [CrossRef] 48 • Can et al. The Effects of Conivaptan on Post-Ischemic Brain Injury and Edema Eurasian J Med 2019; 51(1): 42-8

25. Trabold R, Krieg S, Scholler K, Plesnila N. Role 27. Kapural M, Krizanac-Bengez L, Barnett G, Perl J, 29. Breckwoldt MO, Chen JW, Stangenberg L, et al. of vasopressin V(1a) and V2 receptors for the et al. Serum S-100beta as a possible marker of Tracking the inflammatory response in stroke in vivo development of secondary brain damage after blood-brain barrier disruption. Brain Res 2002; by sensing the enzyme myeloperoxidase. Proc Natl in mice. J Neurotrauma Acad Sci USA 2008; 105(47): 18584-89. [CrossRef] 2008; 25: 1459-65. [CrossRef] 940: 102-4. [CrossRef] 30. Can B, Öz S, Musmul A, et al. Effects of conivap- 26. Glushakova O, Glushakov A, Miller E, Valadka 28. Kanazawa M, Kawamura K, Takahashi T, et A, Hayes R. Biomarkers for acute diagnosis tan and mannitol on serum cytokine levels al. Multiple therapeutic effects of progranulin and management of stroke in neurointensive (TNF-α, IL-15 and IL-35) following bilateral care units. Brain Circulation 2016; 2: 28-47. on experimental acute ischaemic stroke. Brain carotid artery occlusion. Eurasian J Bio Chem Sci [CrossRef] 2015; 138: 1932-48. [CrossRef] 2018; 1: 22-8.