Plasma CRH Level Differece Betwee Wistar Rats Exosed To Acute Stress Due To Predator ORIGINAL ARTICLE PLASMA CORTICOTROPINE RELEASING HORMONE (CRH) LEVEL DIFFERENCE BETWEEN WISTAR RATS EXPOSED TO ACUTE STRESS DUE TO PREDATOR AND TO THE PSYCHOLOGICAL STRESS DEVICE B Hadoko Daeg*, Aalis Wisu Wardhaa**, Aris Widodo***, Hidayat Sujuti***, Karyoo Mitaroem***, Edi Widjajato*** *Widya Madala Catholic Uiversity Surabaya, Idoesia; **Faculty of Veteriary Medicie, Brawijaya Uiversity, Malag, Idoesia; ***Faculty of Medicie, Brawijaya Uiversity, Malag, Idoesia. Abstract Objective: Stress triggers ad causes sychiatric disorders. This study comared stress geerated by differet stressors: a cat as the redator of rats ad a Psychological Stress Device () which was develoed ad modified by the researchers based o the model by Xu ad Rocher. Methods: Twety-eight Wistar rats were simle radomly divided ito oe cotrol grou ad six treatmet grous, each cosistig of 4 rats. Each treatmet grou was idividually exosed to stressor for 30, 60, ad 90. The first three treatmet grous were treated usig the while the other three treatmet grous were treated exosed to the cat. Plasma CRH level was measured usig the ELISA (Cusabio) method. Result: Plasma CRH levels i the rat exosed to stressor usig the raged from 9.89 to 50.22 g/ml, higher tha lasma CRH level i the grous exosed to cat raged from 0.22 to 23.44 g/ml with sigificace level (<0,05). The average of lasma CRH level i the rats exosed to the for 30, 60, ad 90 were 14.83, 28.19, ad 36 resectively. 14 g/ml while i the grous exosed to cats were 11.53, 7.81, ad 4.97 g/ml resectively. The icrease of lasma CRH level had ositive correlatio with the legth of exosure to stressor i the grou treated with the (r=0.895, of <0.05) while lasma CRH level i the grou exosed to cat did ot correlate with the legth of exosure (r=-0.043, >0.05). Coclusio: Plasma CRH level of the rats exosed to stressor usig the was higher ad ositively correlate with the legth of exosure comared to those exosed to cat. ASEAN Joural of Psychiatry, Vol. 16 (2): July December 2015: XX XX. Keywords: Stressor, Acute Stress, Predator, Psychological Stress Device, Plasma CRH Itroductio Acute stress is a coditio where a idividual is exosed to a stressor from a few to several hours [1]. Further, Bhatia et al. (2011) claimed that acute stress o aimal uder exerimet is traslated as their beig exosed to a stressor oce ad by the same kid. I the sychiatric study, both acute ad chroic stress may trigger or cause sychiatric disorders. Oe good examle is the biolar disorder which is a mood disorder iitiated by real sychological stress. The recurrece of biolar disorder is mostly triggered by a sychological stress[2], ad so are other sychiatric disorders. The stressful coditio stimulates aravetricular ucleus (PVN) to release corticotroie releasig hormoe (CRH) which will the affect the body fuctios, immuity system, ad sychological fuctios [1].
Plasma CRH Level Differece Betwee Wistar Rats Exosed To Acute Stress Due To Predator I most studies, the treatmet of exosig exerimetal aimals to stressors does ot differetiate sychological from hysical or social stressors. Bhatia, et al. (2011), i his study rescribes that iducig sychological stresses to rats ca be erformed by exosig them to a cat as their redator, oise, eoatal isolatio, ad light[3]. This study used Psychological Stress Device () which was develoed ad modified by the researchers based o the oe desiged by Xu et al. (1998) ad also utilized by Rocher et al. (2004) i his study. Oce rats exeriece acute stress, they will exhibit sigs of axiety, freezig, iloerectio, defecatig, ad uriatig while beig o the latform [4, 5]. Cats as creatures may ted to be ustable. As a result, it is ot a stable source of stressor. The stressor device, o the other had, is stable ad stadardized. This study examied the differeces i the level of lasma CRH betwee grous of rats exosed to a cat as their redators ad the. Methods Subjects The subjects from the study were 30 Wistar rats obtaied from the Pharmacology Laboratory of the Faculty of Health Sciece of Muhammadiyah Uiversity Malag, Idoesia. All the rats used for this study were males, with their iitial weight ragig from 93 to 146 grams. The rats were acclimatized for 14 days, ket i six lastic cages with the dimesio of 30 cm x 12 cm x 35 cm. Each cage accommodated 5 rats. The cages were wove with strigs, ad hays were laced iside them. The rats were exosed to light ad darkess for each 12 hours a day, amly fed, ad ut i a quiet lace. Whe the rats arrived at the laboratory, each rat was weighed, ad a code was tagged o its tail. The rats were weighed agai o the ext morig before beig give treatmet. Twety-eight out of the 30 rats with the average weight ragig from 119-188 grams were selected. The other two were ot ivolved i the study due to their isufficiet body weight, ad it was oly 28 rats were eeded. The rats were simle radomly divided ito 7 grous by drawig the lottery aer. Each grou cosisted of four rats. The first grou was decided to be the cotrol grou. The secod three grous of rats were exosed to, each grou with the legth of 30, 60, ad 90 cosecutively. The third three grous were exosed to their redator, a cat, each grou with the legth of 30, 60, ad 90 cosecutively. Certificate of Ethics o Aimal Testig ad Research Licese Before this, study was executed, the researchers alied for the certificate of ethics o Aimal testig from the Commissio of Ethics of Health Research of Brawijaya Uiversity, Malag with the ref. umber of 228/EC/KEPK PPDS/05/2013. The licese of research was obtaied from the Pharmacology Laboratory ad the Physiology of the Faculty of Medicie of the resective Uiversity. Treatmet Exosure to Psychological Stress Device () I this study, a latform made of trasaret acrylic with the legth of 20 cm ad width of 21 cm was made. The latform stads o a ole with the height of 100cm from the groud (Figure 1). Figure 1. The used i this research
Plasma CRH Level Differece Betwee Wistar Rats Exosed To Acute Stress Due To Predator I order to make the latform ustable, a ball ad socket joit was laced betwee the ole ad the latform whose thickess had bee adated to the latform, thus eablig the latform to move i all directios with the maximum lea agle of 6 degrees, eough to make the rats stressed out. The ot-so-stee agle also eabled the rats to kee exlorig without fallig dow from the latform. The device is kock-dow i ature. It is made based o Xu et al., 1998 ad Rocher et al. 2004. Each rat was laced o the uder a 60 watt lam. The rats were let to freely exlore ad move o the latform, ad the researchers at the same time observed the sigs of stress, which icluded freezig, iloerectig, defecatig, ad occasioal uriatig that the rats exhibited (Figure 2 ad 3). The treatmets lasted for 30, 60, ad 90. Figure 2. The rat is o the latform of the Urie Faeces Figure 3. The rat is o the latform of the, some amout of urie ad fecal matter was visible because the rat was uder a stressful coditio Soo after each treatmet was fiished withi the laed duratio of time, the rats were take from the latform, ad they were sacrificed by dislocatig their columa vertebra cervicalis. Their abdomes were oeed u to the thorax by a air of scissors. As the heart was sotted, the blood was slowly asirated from it with a 3 ml syrige for about 2 ml. The blood was the laced iside a bottle cotaiig EDTA, labeled accordig to the codificatio, ad stored i the refrigerator for the examiatio of the lasma CRH. A cat as their redator The redator ivolved i this study was a domestic cat ket i a cage with the dimesio of 40 cm X 60 cm X 40 cm. The rats were idividually laced ito a cage with the dimesio of 26 cm X 20 cm X 10cm, ad the the cage i which the rats were idividually laced for 30, 60, ad 90, was ositioed iside the bigger cage where the cat was aroud. (Figure 4).
Plasma CRH Level Differece Betwee Wistar Rats Exosed To Acute Stress Due To Predator Figure 4. The rat i its cage was idividually The sigs of stress the researchers oticed were freezig, iloerectio, defecatig, ad occasioal uriatig. Soo after each treatmet was fiished withi the laed duratio of time, the rats were take from the latform, ad they were sacrificed by dislocatig their columa vertebra cervicalis. Their abdomes were oeed u to the thorax by a air of scissors. As the heart was sotted, the blood was slowly asirated from it with a 3 ml syrige for about 2 ml. The blood was the laced iside a bottle cotaiig EDTA, labeled accordig to the codificatio, ad stored i the refrigerator for the examiatio of the lasma CRH. Results they were just brought to the laboratory. Two rats were excluded from this study for two reasos. First, they were uderweight, adsecod;oly 28 rats were eeded i this study. The result from the aalysis i Table 1 revealed that data of iitial weights, weight after 2 weeks, ad weight gai had ormal distributio i all grous (Shairo-Wilk >0.05). The variatios from the data were homogeous amog the grous (Levee s test >0.05). Data aalysis usig oe-way Aova cofirmed that there were o sigificat differeces i iitial weight, weight after 2 weeks, ad weight gais amog the grous. The Body weight of the Rats The body weight of the rats whe arrivig ad before treatmet icreased comared to whe
Table 1. Comariso of the body weight of the rats amog the grous based o legths of treatmets Legths of Measuremet Iitial weight Weight after 2 weeks Weight gai 30 60 90 Predator(Cat) 30 Predator(Cat) 60 Cat 90 30 60 90 Predator(Cat) 30 Predator(Cat) 60 Predator(Cat) 90 30 60 90 Predator(Cat) 30 Predator (Cat) 60 Predator (Cat) 90 Body Weight (grams) 4 114.75 21.716 97 146 4 104.00 11.518 92 119 4 105.75 7.632 99 116 4 108.75 9.743 101 123 4 116.75 16.460 93 130 4 99.50 30.447 56 127 4 109.75 12.366 98 127 4 144.25 22.882 128 177 4 155.25 22.969 137 188 4 137.50 14.107 123 154 4 139.75 16.820 119 160 4 164.00 17.455 141 179 4 141.00 19.201 120 163 4 149.25 9.811 135 157 4 29.50 12.610 16 46 4 51.25 26.247 18 82 4 31.75 14.385 22 53 4 31.00 18.166 15 54 4 47.25 18.661 22 67 4 41.50 20.632 23 64 4 39.50 8.021 30 49 0.815 0.389 0.510 PlasmaCRH Level The lasma CRH level of the 4 rats i the cotrol grou, the grou which was ot exosed to stressor, raged from 0.67 to 11.11 g/ml. There were three grous, which were exosed to the, ad each cosisted of 4 rats. The lasma CRH level of the first grou which was exosed to stressor for 30 varied, with the lowest 0.22 g/ml ad the highest 12.22 g/ml while the lasma level of the secod grou which was exosed to stressor for 60 varied, with the lowest 22.56 g/ml ad the highest 34.56 g/ml. Fially, the lasma CRH level of the last grou which was exosed to stressor for 90 also varied, with the lowest 0.35 g/ml ad the highest 50.22 g/ml. From the
results, it was clear that the loger the treatmet was give, the higher the lasma level became (r=0.895, <0.05). Furthermore, the last three grous which were exosed to a cat as their redator also cosisted of 4 rats each. The lasma level of the first grou which was exosed to stressor for 30 varied, with the lowest 0.11 g/ml ad the highest 23.44 g/ml while the lasma level of the secod grou which was exosed to stressor for 60 varied, with the lowest 0.56 g/ml ad the highest 20.33 g/ml. Fially, the lasma level of the last grou which was exosed to a stressor for 90 also varied, with the lowest 0.22 g/ml ad the highest 9.44 g/ml. It ca be erceived that the highest lasma CRH level, 23.44 g/ml, was obtaied from the grou which was exosed to a cat for 30, ad the lowest lasma CRH level, 0.11 g/ml, for 90. It ca be summarized that the icrease of lasma level is ot lieary correlated with the legth of treatmet (r=- 0.043, >0.05). The data of lasma CRH level had ormal distributio i all grous (>0.05) ad had homogeous variatios amog all grous (>0.05). The comariso with the CRH level betwee grous exlaied that there were sigificat differeces at least betwee 2 grous (<0.05). Briefly, the result of the aalysis is deicted i the followig diagram. CRH Plasma Level g/ml 40 35 30 25 20 15 10 5 0 36.139 28.194 14.833 4.583 11.528 7.806 4.972 0' 30' 60' 90' Legth of Exosure Cat Legth of Exosure Figure 5. The fluctuatio of the lasma CRH level stimulated by (dark grey) ad a cat as redator (light grey) I figure 5, clearly the icrease with the lasma level stimulated by was lieary correlated to the legth of the exosure, while the icrease of lasma CRH level i the grous exosed to a cat as their redator oly took lace util the 30th miute, makig the chages much less sigificat. The CRH level of the grous exosed to the differed sigificatly alog with the duratios of the treatmet (<0.05). Table 3 reveals that the loger exosure to the, the higher the CRH level. The lasma CRH level of the grous exosed to a cat as their redator did ot icrease sigificatly over legth of treatmets (>0.05). Table 4 exhibits the comariso of the lasmacrh levelof the rats stimulated by a cat as their redator amog the observatio eriods. The lasmacrh level was oly icreasig durig the first 30, ad it was gradually decreasig afterwards. The gradual decrease over the eriods of the treatmets was susected due to the rats beig able to adat themselves with the existece of their redator over the time. Table 5 reveals the comariso with the lasma level of the rats stimulated by the ad a cat as their redator withi the
observatio eriod of 30. The result from the aalysis revealed that there were o sigificat differeces i the lasma level (>0.05) both i the grous that were exosed to the ad to the oes that were exosed to a cat as their redator. Table 6 reveals that the comariso with the lasma CRH level of the rats stimulated by ad a cat as their redator withi the observatio eriod of 60 was sigificatly differet (<0.05). Further; the lasma level was higher i the grous that were exosed to the comared with the oes that were exosed to a cat as their redator. Table 7 reveals the comariso with the lasma level of the rats stimulated by ad a cat as their redator withi the observatio eriod of 90. The result from the aalysis revealed that the lasma level was higher for the grous that were exosed to the comared with the oes that were exosed to a cat as their redator. Tabel 2. The Comariso of lasma CRH Level amog Treatmets 30 60 90 Predator(cat) 30 Predator(cat) 60 Predator(cat) 90 CRH level (g/ml) 4 4.583 4.604 0.6667 11.1111 4 14.833 5.253 9.8889 22.0000 4 28.194 5.733 22.5556 34.5556 4 36.139 10.283 25.5556 50.2222 4 11.528 8.730 2.5556 23.4444 4 7.806 9.091 0.5556 20.3333 4 4.972 3.807 0.2222 9.4444 P <0.001 Table 3. The comariso of the lasma CRH level of the rats stimulated by sychologicalstress device amog grous ad observatio eriods CRH Plasma Level (g/ml) P Plasma CRH Level (g/ml) 4 4.583 4.604 0.6667 11.1111 4 14.833 5.253 9.8889 22.0000 30 4 28.194 5.733 22.5556 34.5556 P <0.001 60 90 4 36.139 10.283 25.5556 50.2222
Table 4. The comariso of the lasma CRH level of the rats stimulated by a cat as their redator amog grous ad observatio eriods Predator(cat) 30 Predator(cat) 60 Predator(cat) 90 Plasma CRH Level (g/ml) 4 4.583 4.604 0.6667 11.1111 4 11.528 8.730 2.5556 23.4444 4 7.806 9.091 0.5556 20.3333 4 4.972 3.807 0.2222 9.4444 0.494 Table 5. The comariso of the lasma CRH level of the rats stimulated by ad a cat as their redator withi the observatio eriod of 30 30 Predator(cat) 30 Plasma CRH Level (g/ml) 4 14.833 5.253 9.8889 22.0000 4 11.528 8.730 2.5556 23.4444 P 0.540 Table 6. The comariso of the lasma CRH level of the ratsstimulated by ad a cat as their redatorwithi the observatio eriod of 60 60 Predator(cat) 60 Plasma CRH Level (g/ml) 4 28.194 5.733 22.5556 34.5556 4 7.806 9.091 0.5556 20.3333 0.009 Table 7. The comariso of the lasma CRH level of the rats stimulated by ad a cat as their redator withi the observatio eriod of 90 90 Predator(Cat) 90 Plasma CRH Level (g/ml) 4 36.139 10.283 25.5556 50.2222 4 4.972 3.807 0.2222 9.4444 0.001 Discussio This exerimet aims at comarig the chagig of the level of CRH i the lasma of the rats exosed by the sychological stress device (hece: ) ad the oes by redator, i this study a cat. Cat is a atural redator of rats. Therefore, the behavior of a cat as a livig beig, esecially its aggressiveess is tremedously iflueced by the surroudig as well as by the ier emotio of the cat itself. As a result, its behavior is usteady. O the other had, the stressor roduced by the is steady, measurable, adjustable, ad stadardized. Has Selye i his cocet Geeral Adatatio Sydrome defies stress as a coditio where there is a homeostatic disrutio [6]. Furthermore, stress is defied as a chai of occurreces, icludig stimulus (stressor) which reciitates the reactio from the brai (the ercetio ad rocessig of stress). This sequece activates the hysical system of
fight-or-flight" i the body, which is a ormal resose to stress [1]. Basically, both defiitios refer to the struggle of livig beigs to adat themselves to their eviromet i order to survive. Previous researches that iduced stress to tested aimals geerally make use of the term of stress, but they do ot further differetiate whether it is sychological, hysical, or social stress [7, 8]. I reality, the first oset, relase ad recurret of sychiatric disorder are likely to be receded by a sychological stressor [2] although hysical stress ca also trigger sychiatric disorder. The stress which was iduced by otraumatic hysical restrai o the rats beig tested icreased the CRH level (8). However, it is quite far from beig a real sychological stressor i the real sese, although there was a factor of hoelessess. The result from the aalysis reveals that the rats i all grous had a ormal distributio of body weight both i the begiig ad withi two weeks (Shairo- Wilk >0.05). The gaiig of the body weight was homogeous amog the grous (Levee s test >0.05). The oe-way Aova aalysis also cofirmed the result that there was o sigificat chage i the body weight amog the grous both i the begiig ad withi two weeks. A dro i body weight ad aetite was idetified i the tested aimals which had bee sufferig from deressio [7]. The rats, havig bee acclimatized i the laboratory for two weeks, gaied more weight, sigifyig that oe of the rats used for this exerimet exerieced stress rior to the treatmet. The weight icrease betwee the two grous, however, was ot sigificatly differet, ad it. Cosequetly, did ot ifluece the rise of the CRH level i the lasma. Previous researchers have reseted the evidece that a stressful coditio icreases the CRH level [9], ad the oe by Eosito et al (2001) cofirmed a similar result. O the other words, stress is closely related with the icrease of CRH level [8]. All the rats exosed to visually showed more severe sigs of stress; beig axious, freezig, defecatig, or uriatig [5, 4] (Figure 3) comared to the other grous treated usig a cat as their redator. Those sigs of stress were i accordace with the sigificat rogressive rise to the CRH level i the lasma of the grous exosed to a stressor geerated by the comared with the oe exosed to a stressor by itroducig a cat as their redator. The CRH level i the lasma o the grous which were iduced to stress by a cat as their atural redator oly ket risig withi 30, ad a cotiuous declie was foud afterwards. There were two reasos uderlyig this heomeo. First, the rats must have adated themselves with the resece of the cat as their redator, ad secod the aggressiveess of the cat as a creature teded to be ustable. Meawhile, the CRH level i the lasma o the grous which were iduced to stress by ket risig startig from 30, 60, to 90 i a cosistet ad liear maer sice as a mechaic device was stable, adjustable, ad measurable (Figure 1). The coditio of acute stress activates the HPA axis which the releases CRH [8]. I this research, it ca be cocluded the level of CRH rises sigificatly i a liear maer with the treatmet duratio of 30, 60, ad 90. Nakamura i his iteratioal worksho about Iteratioal Aimal Research Regulatio: Imacto Neurosciece Research: NCBI Bookshelf rooses about aimal welfare, ad miimizig ai ad distress [10]. The cliical imlicatio from this study is to kow the relatioshi of sychological stress ad athosychobiology of sychiatric disorders. With the uderstadig of this relatioshi, revetio ad treatmet would be better develoed. The study limitatio is we caot use various secies of exerimetal aimals (Rats ad Cats), because it will be more comlicated ad require lots of studies, although there is a ossibility that the result will be differet if usig various secies. For the, rats laced uo the latform i this study sometimes fall from the latform because it caot avoid the rats from jumig. The researcher recommeds the discrimiatio of hysical, sychological, ad social stressors as well as fair aimal treatmet. All researchers, esecially those ivolvig aimals
i a direct maer, should ay a close attetio to their welfare as well as miimizig their ai ad distress. I coclusio, coclusio, the body weight of the rats uder study was ot sigificat to the risig level of CRH i the lasma of the rats. The rise of CRH lasma i the grous iduced to stress by tured out to be ot oly higher but also more stable comared to both the cotrol grous, ad the oes iduced to stress by a cat as their redator. The rise of CRH lasma i the grous iduced to stress by was liear alog with the legth of the exosure. Refereces 1. Dhabhar FS ad McEwe BS. Bidirectioal Effects of Stress o Immue Fuctio: Possible Exlaatios for Salubrious as Well as Harmful Effects i AderR, ed: Psychoeuroimmuology, Fourth Editio, Vol I,Book 2, Chater 34, Burligto, MA, USA: Elsevier Academic Press. 2007; 723-60. 2. Vieta E. Maagig Biolar i Cliical Practice, 2d Editio, Curret Medicie Grou Ltd., Lodo. 2009. 3. Bhatia N, Maiti PP, Choudary A, Tuli A, Masih D, Kha MMU, Ara T, Jaggi AS. Aimal Model of Stress, Iteratioal Joural of Pharmaceutical Scieces ad Research. 2011; 2(5): 1147-55. 4. Xu L, Holscher C, Awyl R, Rowa MJ. Glucocorticoid recetor ad rotei/rna sythesis-deedet mechaism uderlie the cotrol of syatic lasticity by stress, Proc Natl Acad Sci USA. 1998; 95:3204-08. 5. Rocher C, Seddig M, Muoz C, Jay TM. Acute Stress-iduced Chages i Hiocamal /Prefrotal Circuits i Rats: Effects of Atideressats, Cerebral Cortex. 2004; 14:224-229. 6. Sutato, W, & Kloet, ER de. The Use of Various Aimal Models i The Study of Stress ad Stress-related Pheomea, Laboratory Aimal. 1994; 28: 293-306. 7. Duma, CH. Model of Deressio, Vitamis ad Hormoes, Chater 1, Vol 82, Deartmet of Psychiatry, Yale Uiversity, Coecticut Metal Health Ceter, New Have, Coecticut, USA. 2010; 1-21. 8. Esosito, P, Gheorghe, D, Kadere, K, Pag, X, Coolly, R, Jacobso, S, Theoharides, TC. Acute Stress Icreases Permeability of the Blood- Brai Barrier through activatio of Brai Mast Cells, USA, Brai Research. 2001; 888: 117-27. 9. Theoharides, TC, Roziecki, JJ, Sahagia, G, Jacobso, S, Kemuraj, D, Coti, P, ad Kalogeromitros, D. Imact of Stress ad Mast Cells o Brai Metastases, ClicalKey, Elsevier. 2008; 12-15Z: 205(1-2): 1-7. 10. Nakamura, R.Natioal Academy of Scieces, Iteratioal Aimal Research Regulatio: Imact o Neuroscieces Research: Worksho summary, NCBI Bookshelf, Natioal Academy of Sciece, Natioal Academic Press (US), Washigto (DC). 2012. Corresodig author: B. Hadoko Daeg, Dr., S.KJ(K), Widya Madala Catholic Uiversity Surabaya, Raya Kalisari Selata 1,Tower Barat, Lt 8, Pakuwo City, Surabaya, Idoesia. Email: bhadokodaeg@yahoo.com Received: 29 August 2014 Acceted: 7 July 2015