Expression Of Eat-1 MRNA In The Adult Rat Spinal Cord Under Normal Condition And After Different Types Of Axotomy

Expression of Eat-1 mRNA in the adult rat spinal cord under normal condition and after different types of axotomyObjectiveEta-1 (Osteopontin, OPN) is a secretory adhesive glycoprotein that was originally isolated from the bone and subsequently found in the kidney, inner ear, and various other tissues. The widespread distribution of Eta-1 suggests that it has multiple functions, but its precise functions are not fully understood.Recently, the expression of Eta-1 has been observed in neurons in the olfactory bulb and the brain stem; it has also been identified in the activated microglia after focal brain ischemia, suggesting that Eta-lcould also play a role in the central nervous system(CNS). However, the distribution of Eta-1 in another component of CNS, the spinal cord, has not been clarified.Spinal nerve axotomy presents a difficult problem that is commonly encountered by orthopedic surgeons in their clinical practice. Its pathological changes and molecular mechanism affecting axon regeneration after injury are not fully understood.The fate of spinal motoneurons after axotomy is dependent on site of the lesion. Thus, in the adult rat, no motoneurons are lost following midthigh sciatic nerve transection, whereas a spinal root avulsion lesion, which occurs at the interface of CNS and PNS and accomplishes a proximal axotomy of motoneurons, is followed by an extensive cell death (up to 80%) among affected motoneurons.The CNS and PNS differ fundamentally in their response to axonal injury. In the PNS, transected axons can regrow and extend beyond the site of injury, achieving at least partial functional recovery. In contrast, axotomized fiber tracts in the CNS exhibit abortive sprouting but do not succeed in crossing the injury site into the distal fiber tract. It has been shown that the tissue specific environment may induce differential molecular programming of lesion-associated macrophages, which in turn influences the extent of axon regeneration after injury.The matricellular glycoprotein Eta-1 is among the most abundant secretory products of lesion-associated macrophages, It has been detected in a number of injury models in brain, heart, and other peripheral organ systems. Functionally, Eta-1 provides a chemotactic stimulus for macrophages, fibroblasts, and astrocytes, and mediates potent immunoregulatory effects in inflammatory and autoimmune conditions.There has been, however, no report concerning Eta-1 mRNA expression following different type axotomy.In the present study, the distribution of Eta-lmRNA in the adult rat spinal cord has been investigated by in situ hybridization and fluorescence immunohistochemistry. Then, we used the spinal root avulsion and sciatic nerve transection models and investigated the expression of Eta-1 after different type of axotomy to elucidate its possible role in axon injury and axon regeneration.Materials and Methods1. Normal tissue preparationTen Eight-week-old male Wistar rats (average weight 200g) were used for all the experiments. Animals were anesthetized deeply with pentobarbital. For histological evaluation, the animals were perfused intracardially with ice-cold saline, followed by perfusion of ice-cold 4% paraformaldehyde in PBS (pH 7.4). Tissue blocks containing C5-7, T8-10, L2-4 spinal segments were collected, postfixed in the same fixative overnight at 4℃, and cryoprotected in 20% sucrose in PBS overnight. The tissue blocks were freeze-mounted on a holder, cut into 12 m sections with a cryostat and placed on poly-L-lysine-coated glass slides. 2. In situ hybridizationIn situ hybridization was performed to detect the expression of Eta-1.3. Double staining by in situ hybridization and fluorescence immunohistochemistryAfter in situ hybridization, sections were washed with PBS for 5 min, and then reacted overnight at 4℃with monoclonal mouse anti-NeuN as the primary antibody. After washing with PBS 3 more times for 10 min each; the sections were reacted with an Alexa Fluor 488-conjugated goat anti-mouse IgG antibody and mounted on slides with fluorescent mounting medium.4. Animal model of spinal root avulsionEight-week-old male Wistar rats (average weight 200g; 40 rats) were used for all the experiments. Extravertebral avulsion of the L3 and L4 nerves was performed according to the method of Koliatsos et al. with slight modifications.5. Animal model of sciatic nerve transectionEight-week-old male Wistar rats (average weight 200g; 40 rats) were used. Left sciatic nerves were exposed at mid-thigh level and transected with a fine pair of scissors. The proximal transected nerve stumps were tied with surgical silk and kept separated.6. Tissue preparation from axotomized ratsThe animals were sacrificed at 1, 3, 7, 14, or 21 days postlesion. For Northern blot analysis, the L2-L5 segments of the spinal cord were removed and frozen immediately in liquid nitrogen. For histological evaluation, the animals were perfused intracardially with ice-cold saline, followed by perfusion of ice-cold 4% paraformaldehyde in PBS. Tissue blocks containing the L3 and L4 spinal segments and the left sciatic nerve were collected, postfixed in the same fixative overnight at 4℃, and cryoprotected in 20% sucrose in PBS overnight. The tissue blocks were freeze-mounted on a holder, cut into 12μm sections with a cryostat and placed on poly-L-lysine-coated glass slides.7. Preparation of digoxigenin (DIG)-labeled Eta-1 probes for Northern blot analysis8. Northern blot analysis9. Nissl staining:Sections were stained with cresyl violet.10. In situ hybridization11. ImmunohistochemistryImmunohistochemistry was performed as described previously. The primary antibodies were mouse anti-neuronal nuclei, anti-human Eta-1 monoclonal antibodies, and a mouse anti-rat CD11b monoclonal antibody (OX42). To identify the two tissue antigens in a single section, the double-staining method described by Vandesande was used with some modifications.12. Double staining by in situ hybridization and fluorescence immunohistochemistry13. Cell count of NeuN-positive neurons, OX42-reactive macrophages, and Eta-1 expressing cells.14. Statistical analysisDifferences between groups were evaluated by ANOVA, followed by the Fisher protected least significant difference test. p values less than 0.05 were considered to be significant. Results1. Eta-1 mRNA expression in normal spinal cordResult of in situ hybridization studies displayed the expression of Eta-lmRNA in motoneurons, which were identified by their morphology, but no signal in the posterior horn. Eta-lexpression in motoneurons was further confirmed by combined labeling with fluorescence immunohistochemistry for NeuN and in situ hybridization for Eta-1.2. Northern blot analysisA low level of Eta-1mRNA expression was detected in the normal spinal cord. After avulsion, levels of Eta-1mRNA were upregulated from day 1. The highest levels, 1.8 times higher than that in the normal spinal cord (p＜0.05), were observed from days 1 to 3 after avulsion.3. Loss of motoneurons following spinal root avulsionThe decrease of motoneurons in the ipsilateral (avulsion side) anterior horn was evident from 3 days postlesion, Cell loss dramatically increased with time, and by day 21, more than 50% of motoneurons on the ipsilateral side had degenerated.4. Eta-1 positive motoneurons and astrocytes in control and avulsed ratsThree days after injury, the level of hybridization signal in motoneurons was elevated. At 21 days, Eta-1mRNA expression levels had returned to the normal level in motoneurons. There was no Eta-1mRNA expression in the posterior hom of control rats; however, Eta-1mRNA expression was observed in small ceils in the posterior horn 3 days after injury and had disappeared at 21 days. by combining fluorescence immunohistochemistry and in situ hybridization for Eta-1, these Eta-1-expressing cells were confirmed to be motoneurons and astrocytes.5. Eta-1 expression in macrophages following avulsionIn the uninjured tissue, many ramified OX-42-reactive cells were present. However, after avulsion, numerous strongly OX-42-labeled macrophages with small, round, and amoeboid morphology appeared from day 1 and were most abundant on day 3. A subpopulation of these cells was colocalized with intense Eta-limmunoreactivity, the number of which increased from 1 day postlesion to a peak on day 3.6. Macrophages in sciatic nerve transection do not express Eta-1In the sciatic nerve, the transaction site was densely infiltrated by OX-42~+ macrophages. In contrast to spinal root avulsion, at none of the time points (3d-21d after injury) was Eta-1 induction evident. Motoneuron loss in the anterior horn of spinal cord was not significant; and the alteration of Eta-1 expression by motoneurons was not significant, either.7. Quantitative analysis of Eta-1-positive neurons and macrophages following avulsionWe compared the numbers of Eta-1-expressing neurons (NeuN-positive) in the anterior horn and Eta-1-expressing macrophages (OX-42-reactive) in the posterior horn between the ipsilateral and contralateral sides at the avulsion level on day 3. Although the number of NeuN-positive neurons was reduced on the injured side, the number of Eta-1-expressing cells had increased. In the posterior horn, the numbers of OX-42-reactive macrophages and Eta-1-expressing cells increased simultaneously compared to the contralateral side.ConclusionThe distribution of Eta-1mRNA in motor-related areas (anterior horn) in whole spinal cord was first demonstrated in our study, and it suggests other specialized functions of Eta-1under normal condition in the adult spinal cord. However, the exact role of Eta-lin the normal spinal cord remains to be further studied.Upregulation of Eta-lexpression was observed in motoneurons, astrocytes, and activated macrophages following spinal root avulsion. While macrophages in sciatic nerve transection do not express Eta-1. We suggest that Eta-1has multiple different functions in the nervous system:①. in the inflammatory response, mediated by glia and possibly neurons;②. may contribute to the non-permissive nature of the adult CNS preventing axonal regeneration following injury and③. in the protection of neurons from iNOS-mediated degeneration.