| Abstract | Cilj: Cilj ovog istraživanja bio je utvrditi izražaj i raspored koneksina (Cx) 37, 40, 43 i 45 u kralježničnoj moždini štakora te njihove eventualne promjene u kratkotrajnoj šećernoj bolesti izazvanoj aplikacijom streptozotocina u štakora.
Materijal i metode: Deset muških štakora soja Sprague-Dawley podijeljeno je u dvije skupine, kontrolna skupina (K) i dijabetična (DM). U dijabetičnoj skupini štakorima je aplicirano intraperitonealno 55 mg/kg streptozotocina (STZ), koji je otopljen u citratnom puferu (pH 4.5), kako bi se inducirala šećerna bolest tipa 1, a štakori koji su korišteni kao kontrola dobili su čisti citratni pufer. Životinje su eutanazirane nakon dva tjedna te im je uklonjena kralježnična moždina. Tkivo je uklopljeno u parafin. Rezovi tkiva obojani su imunohistokemijski korištenjem protutijela protiv Cx37, Cx40, Cx43 i Cx45. Uzorci su pregledani pomoću epifluorescencijskog mikroskopa. Mikrofotografije rezova su analizirane pomoću Adobe Photoshop i Image J programa. Analizirano je područje dorzalnog roga i područje oko (uključivo) centralnog kanala. Rezultati su statistički obrađeni korištenjem Mann Whitney testa ili Kruskal-Wallis testa s Dunn-ovim post-hoc testom za višestruke usporedbe, pri čemu je p<0.05 smatrano značajnom razlikom.
Rezultati: Na histološkim rezovima kralježnične moždine štakora pronašli smo imunoreaktivnost Cx37 i Cx40 u neuronima, dok je izražaj Cx43 bio najjači u okolnoj gliji. Cx45 pronašli smo u bijeloj tvari, kao i oko mijeliniziranih vlakana, gdje je uočen i Cx37. U krvnim žilama pronađeni su Cx37, Cx40 i Cx43, dok su u ependimskim stanicama centralnog kanala bila izražena sva četiri istraživana koneksina. Usporedbom izražaja pojedinih koneksina, utvrdili smo najviši izražaj Cx43, značajno više u odnosu na sve ostale istraživane koneksine, kako u kontrolnoj, tako i u dijabetičnoj skupini štakora; nakon čega slijedi izražaj Cx40, a najmanje su u kralježničnoj moždini štakora zastupljeni Cx37 i Cx45. Analizom izražaja koneksina u području dorzalnog roga uočeno je statistički značajno smanjenje izražaja Cx40 u skupini DM u odnosu na K skupinu (p<0.01). U području (uključivo) centralnog kanala uočen je značajan porast izražaja Cx37 i Cx43 (p<0.05) i pad izražaja Cx40 (p<0.01). Izražaj Cx45 nije se značajno razlikovao u DM u usporedbi s K skupinom.
Zaključak: Koneksin 37 i Cx40 su u kralježničnoj moždini štakora prvenstveno izraženi u neuronima, dok je Cx43 prvenstveno izražen u astrocitima i mikrogliji, a Cx45 u oligodendrogliji. Eksperimentalni DM1 u štakora u početnoj fazi uzrokuje poremećaj izražaja različitih tipova koneksina, pri čemu se izražaj Cx40 smanjuje, a izražaj Cx43 i Cx37 raste. Promjene izražaja koneksina koje smo utvrdili u kralježničnoj moždini dijabetičnih štakora mogle bi imati značajnu ulogu u procesima maladaptacije somato- i viscerosenzornih puteva izazvanim dijabetesom, kao i utjecati na reaktivnost kralježnične moždine na potencijalnu ozljedu. |
| Abstract (english) | Objective: The aim of this study was to determine the expression and distribution of connexins (Cx) 37, 40, 43 and 45 in the spinal cord of rats and their possible changes in short-term diabetes caused by the application of streptozotocin in rats.
Materials and methods: Ten male Sprague-Dawley rats were divided into two groups, control group (K) and diabetic (DM). In the diabetic group, rats were administered intraperitoneally 55 mg/kg streptozotocin (STZ), dissolved in citrate buffer (pH 4.5), to induce type 1 diabetes, and rats used as controls received pure citrate buffer. The animals were euthanized after two weeks and their spinal cord was removed. The tissue is embedded in paraffin. Tissue sections were stained immunohistochemically using antibodies against Cx37, Cx40, Cx43, and Cx45. The samples were examined using an epifluorescence microscope. Photomicrographs were analyzed using Adobe Photoshop and Image J. The area of the dorsal horn and the area around (inclusively) the central canal were analyzed. Results were statistically processed using the Mann Whitney test or the Kruskal-Wallis test with the Dunn post-hoc test for multiple comparisons, with p <0.05 being considered a significant difference.
The results: On histological sections of the spinal cord of rats, we found the immunoreactivity of Cx37 and Cx40 in neurons, while the expression of Cx43 was strongest in the surrounding glia. Cx45 was found in white matter as well as around myelinated fibers, where Cx37 was also observed. Cx37, Cx40, and Cx43 were found in blood vessels, while all four investigated connexins were expressed in ependymal cells of the central canal. By comparing the expression of individual connexins, we found the highest expression of Cx43, significantly higher than all other studied connexins, both in the control and diabetic groups of rats; followed by the expression Cx40, and the least represented in the spinal cord of rats were Cx37 and Cx45. Analysis of connexin expression in the dorsal horn area showed a statistically significant decrease in Cx40 expression in the DM group compared to the K group (p <0.01). In the area (inclusive) of the central canal, a significant increase in the expression of Cx37 and Cx43 (p <0.05) and a decrease in the expression of Cx40 (p <0.01) were observed. The expression of Cx45 did not differ significantly in DM compared to the K group.
Conclusion: Connexin 37 and Cx40 are primarily expressed in neurons in the spinal cord of rats, while Cx43 is primarily expressed in astrocytes and microglia, and Cx45 in oligodendroglia. Experimental DM1 in rats in the initial stage causes a disturbance in the expression of different types of connexins, with the expression of Cx40 decreasing and the expression of Cx43 and Cx37 increasing. Changes in connexin expression found in the spinal cord of diabetic rats could play a significant role in the processes of maladaptation of somato- and viscerosensory pathways caused by diabetes, as well as affect the reactivity of the spinal cord to potential injury. |
