The use of infrared technology as a novel approach for studies with female laboratory animals

Aim To determine the changes in skin temperature and brown adipose tissue (BAT) activity throughout the estrous cycle as well as the regularity of the estrous cycle in mice. Methods We assessed the differences in the duration of the estrous cycle and its phases between 3- and 8-monthold female mice (n=18). Skin temperature and BAT activity were measured by infrared technology and compared with human menstrual cycle. Results Young and old female mice did not differ significantly in the estrous cycle length. However, young animals had longer diestrus and shorter proestrus phase. In contrast with women, mice showed age-dependent changes in body temperature and BAT activity during the estrus cycle. Conclusion Establishing the pattern of temperature and BAT activity changes could be used to determine the estrous cycle phase before performing experiments without disturbing the animal. However, since the regulation of BAT activity during the estrous cycle was age-dependent, very complex, and varied significantly from women, further studies are needed to develop a non-invasive method for determining the phase of the estrous cycle

Expression of guanylate cyclase C in human prefrontal cortex depends on sex and feeding status

IntroductionGuanylate cyclase C (GC-C) has been detected in the rodent brain in neurons of the cerebral cortex, amygdala, midbrain, hypothalamus, and cerebellum.MethodsIn this study we determined GC-C protein expression in Brodmann areas (BA) 9, BA10, BA11, and BA32 of the human prefrontal cortex involved in regulation of feeding behavior, as well as in the cerebellar cortex, arcuate nucleus of hypothalamus and substantia nigra in brain samples of human 21 male and 13 female brains by ELISA with postmortem delay < 24 h.ResultsGC-C was found in all tested brain areas and it was expressed in neurons of the third cortical layer of BA9. The regulation of GC-C expression by feeding was found in male BA11 and BA10-M, where GC-C expression was in negative correlation to the volume of stomach content during autopsy. In female BA11 there was no correlation detected, while in BA10-M there was even positive correlation. This suggests sex differences in GC-C expression regulation in BA11 and BA10-M. The amount of GC-C was higher in female BA9 only when the death occurred shortly after a meal, while expression of GC-C was higher in BA10-O only when the stomach was empty. The expression of GC-C in female hypothalamus was lower when compared to male hypothalamus only when the stomach was full, suggesting possibly lower satiety effects of GC-C agonists in women.DiscussionThese results point toward the possible role of GC-C in regulation of feeding behavior. Since, this is first study of GC-C regulation and its possible function in prefrontal cortex, to determine exact role of GC-C in different region of prefrontal cortex, especially in humans, need further studies

Uloga urogvanilina u regulaciji prijenosa iona u žlijezdama slinovnicama

Objective of work: Guanylin peptides are considered to be the only intrinsic regulators of salivary glands secretion. Therefore, the aim of this study is to determine the effects of systemic uroguanylin (UGN) of the salivary flow and ion composition as well as if those effects include activation of guanylate cyclase C (GC-C). Materials and Methods: This study was conducted on 7 months old C57Bl6NCrl (wild type, WT) and GC-C knockout (KO) mice. Salivary flow rate and ion composition were determined after pilocarpine stimulation with UGN (30 µg/animal) or saline i.p. application. The expression of mRNA for AQPs, NHEs, NBCn1, Slc26a3/a6 and CFTR were determined by qPCR in submandibular salivary glands. Results: When applied i.p., UGN decreases the pilocarpine stimulated saliva flow rate and increased concentration of Na + , H + and Cl -. In GC-C KO mice, UGN shows no effect on saliva flow rate, while the concentrations of Na + , H + and Cl -are the same in GC-C KO littermates when compared to WT mice. UGN increased expression of Slc26a6 while in GC-C KO mice Slc26a6 had a higher expression when compared to WT mice, suggesting involvement of GC-C independent signalling pathway for UGN. The difference in Slc26a6 in GC-C KO mice is not unique for salivary glands because it was found also in duodenum and kidney cortex. Conclusions: The effects of UGN via basolateral membrane of salivary glands cells have not been considered up to date. In our study, UGN, when applied i.p., decreased salivary flow rate, pH, and changed composition of other ions. Therefore, plasma UGN an hour after a meal could have physiological and pathological importance (development of cavities, inflammations or demineralisations) and inhibition of systemic UGN effects could be consider as a new approach in treatment of those conditions.Cilj rada: Gvanilinski peptidi smatraju se isključivo unutarnjim regulatorima lučenja žlijezda slinovnica. Stoga je cilj ovog istraživanja utvrditi učinke sistemskog urogvanilina (UGN) na protok sline i njen ionski sastav te uključuju li ti učinci aktivaciju gvanilat ciklaze C (GC-C). Materijali i metode: Ovo istraživanje je provedeno na 7 mjeseci starim C57Bl6NCrl (divlji tip, WT) miševima te miševima kojima nedostaje GC-C (GC-C KO). Količina stvorene sline i njen ionski sastav određeni su nakon stimulacije pilokarpinom uz primjenu UGN-a (30 µg/životinji) ili fiziološke otopine i.p.. Izražaj mRNA (glasničke RNK) za AQP-e, NHE, NBCn1, Slc26a3/a6 i CFTR određena je qPCR-om u submandibularnim žlijezdama slinovnicama. Rezultati: Kada se na UGN primijeni i.p., dolazi do smanjenja brzine protoka sline stimulirane pilokarpinom i povećava koncentracija Na + , H + i Cl -. U GC-C KO miševa, UGN nema učinak na brzinu protoka sline, dok su koncentracije Na + , H + i Cl -iste u slini GC-C KO miševa u usporedbi sa WT miševima iz istog legla. UGN je povećao izražaj Slc26a6 dok je i kod GC-C KO miševa Slc26a6 imao veći izražaj u odnosu na WT miševe, što pretpostavlja uključenost GC-C neovisnog signalnog puta za UGN. Razlika u Slc26a6 kod GC-C KO miševa nije jedinstvena za žlijezde slinovnice jer je također pronađena u duodenumu i u bubrežnoj kori. Zaključci: Učinci UGN-a preko bazolateralne membrane stanica žlijezda slinovnica do danas nisu razmatrani. U našoj studiji, UGN, kada se primijeni i.p., smanjuje brzinu protoka sline, pH i mijenja ionski sastav sline. Stoga bi UGN u plazmi sat vremena nakon obroka mogao imati fiziološku i patološku važnost (razvoj karijesa, upale ili demineralizacije), a inhibicija sistemskih učinaka UGN-a mogla bi se smatrati novim pristupom u liječenju tih stanja

The use of infrared technology as a novel approach for studies with female laboratory animals

Aim To determine the changes in skin temperature and brown adipose tissue (BAT) activity throughout the estrous cycle as well as the regularity of the estrous cycle in mice. ----- Methods We assessed the differences in the duration of the estrous cycle and its phases between 3- and 8-month-old female mice (n = 18). Skin temperature and BAT activity were measured by infrared technology and compared with human menstrual cycle. ----- Results Young and old female mice did not differ significantly in the estrous cycle length. However, young animals had longer diestrus and shorter proestrus phase. In contrast with women, mice showed age-dependent changes in body temperature and BAT activity during the estrus cycle. ----- Conclusion Establishing the pattern of temperature and BAT activity changes could be used to determine the estrous cycle phase before performing experiments without disturbing the animal. However, since the regulation of BAT activity during the estrous cycle was age-dependent, very complex, and varied significantly from women, further studies are needed to develop a non-invasive method for determining the phase of the estrous cycle

Uroguanylin increases Ca2+ concentration in astrocytes via guanylate cyclase C-independent signaling pathway

Aim: To investigate the cyclic guanosine monophosphate (cGMP)/guanylate cyclase C (GC-C)-independent signaling pathway in astrocytes, which are a suitable model due to their lack of GC-C expression. ----- Methods: Patch clamp was performed and intracellular Ca2+ concentrations and pH were measured in primary astrocyte cultures and brain slices of wild type (WT) and GC-C knockout (KO) mice. The function of GC-C-independent signaling pathway in the cerebellum was determined by behavior tests in uroguanylin (UGN) KO and GC-C KO mice. ----- Results: We showed for the first time that UGN changed intracellular Ca2+ levels in different brain regions of the mouse. In addition to the midbrain and hypothalamus, GC-C was expressed in the cerebral and cerebellar cortex. The presence of two signaling pathways in the cerebellum (UGN hyperpolarized Purkinje cells via GC-C and increased intracellular Ca2+ concentration in astrocytes) led to a different motoric function in GC-C KO and UGN KO mice, probably via different regulation of intracellular pH in astrocytes. ----- Conclusion: The UGN effects on astrocytes via a Ca2+-dependent signaling pathway could be involved in the modulation of neuronal activity

The Role of Uroguanylin in Regulation of Ion Transport in Salivary Glands

Objective of work: Guanylin peptides are considered to be the only intrinsic regulators of salivary glands secretion. Therefore, the aim of this study is to determine the effects of systemic uroguanylin (UGN) of the salivary flow and ion composition as well as if those effects include activation of guanylate cyclase C (GC-C). Materials and Methods: This study was conducted on 7 months old C57Bl6NCrl (wild type, WT) and GC-C knockout (KO) mice. Salivary flow rate and ion composition were determined after pilocarpine stimulation with UGN (30 µg/animal) or saline i.p. application. The expression of mRNA for AQPs, NHEs, NBCn1, Slc26a3/a6 and CFTR were determined by qPCR in submandibular salivary glands. Results: When applied i.p., UGN decreases the pilocarpine stimulated saliva flow rate and increased concentration of Na + , H + and Cl -. In GC-C KO mice, UGN shows no effect on saliva flow rate, while the concentrations of Na + , H + and Cl -are the same in GC-C KO littermates when compared to WT mice. UGN increased expression of Slc26a6 while in GC-C KO mice Slc26a6 had a higher expression when compared to WT mice, suggesting involvement of GC-C independent signalling pathway for UGN. The difference in Slc26a6 in GC-C KO mice is not unique for salivary glands because it was found also in duodenum and kidney cortex. Conclusions: The effects of UGN via basolateral membrane of salivary glands cells have not been considered up to date. In our study, UGN, when applied i.p., decreased salivary flow rate, pH, and changed composition of other ions. Therefore, plasma UGN an hour after a meal could have physiological and pathological importance (development of cavities, inflammations or demineralisations) and inhibition of systemic UGN effects could be consider as a new approach in treatment of those conditions

Role of uroguanylin's signalling pathway in the development of ischaemic stroke

Stroke is one of the leading causes of mortality and disability worldwide. By affecting bradykinin function, activation of guanylate cyclase (GC)-A has been shown to have a neuroprotective effect after ischaemic stroke, whereas the same has not been confirmed for GC-B; therefore, we aimed to determine the possible role of GC-C and its agonist, uroguanylin (UGN), in the development of stroke. In this study, middle cerebral artery occlusion (MCAO) was performed on wild-type (WT), GC-C KO and UGN KO mice. MR images were acquired before and 24 h after MCAO. On brain slices 48 h after MCAO, the Ca2+ response to UGN stimulation was recorded. Our results showed that the absence of GC-C in GC-C KO mice resulted in the development of smaller ischaemic lesions compared with WT littermates, which is an opposite effect compared with the effects of GC-A agonists on brain lesions. WT and UGN KO animals showed a stronger Ca2+ response upon UGN stimulation in astrocytes of the peri-ischaemic cerebral cortex compared with the same cortical region of the unaffected contralateral hemisphere. This stronger activation was not observed in GC-C KO animals, which may be the reason for smaller lesion development in GC-C KO mice. The reason why GC-C might affect Ca2+ signalling in peri-ischaemic astrocytes is that GC-C is expressed in these cells after MCAO, whereas under normoxic conditions, it is expressed mainly in cortical neurons. Stronger activation of the Ca2+ -dependent signalling pathway could lead to the stronger activation of the Na+ /H+ exchanger, tissue acidification and neuronal death

In relation to NO-System, Stable Pentadecapeptide BPC 157 Counteracts Lidocaine-Induced Adverse Effects in Rats and Depolarisation In Vitro

Recently, the pentadecapeptide BPC 157-induced counteraction of bupivacaine cardiotoxicity has been reported. Medication includes (i) lidocaine-induced local anesthesia via intraplantar application and axillary and spinal (L4-L5) intrathecal block, (ii) lidocaine-induced arrhythmias, (iii) convulsions, and (iv) lidocaine-induced HEK293 cell depolarisation. BPC 157 applications (intraplantar, intraperitoneal, and intragastric) were given (i) immediately after lidocaine, (ii) 10 min after, or (iii) 5 min before. The BPC 157/NO-system relationship was verified with NO-agents, the NOS-blocker L-NAME and the NOS-substrate L-arginine, given alone and/or together, in axillary and spinal intrathecal blocks. BPC 157 applied immediately after lidocaine or 5 min before the application of lidocaine considerably ameliorated plantar presentation. BPC 157 medication considerably counteracted lidocaine-induced limb function failure; L-NAME was counteracted; L-arginine exhibited counteraction when given immediately after lidocaine, but prolongation was seen when given later. Given together, prophylactically or therapeutically, L-NAME and L-arginine (L-NAME + L-arginine) counteracted the other’s response. BPC 157 maintained its original response when given together with L-NAME or L-arginine. When BPC 157 was given together with L-NAME and L-arginine, its original response reappeared. BPC 157 antagonised the lidocaine-induced bradycardia and eliminated tonic-clonic convulsions. Also, BPC 157 counteracted the lidocaine-induced depolarisation of HEK293 cells. Thus, BPC 157 has antidote activity in its own right against lidocaine and local anesthetics