Neurol Sci DOI 10.1007/s10072-014-1840-3

ORIGINAL ARTICLE

Oral administration of inosine promotes recovery after experimental spinal cord injury in rat Maria Kuricova • Valent Ledecky • Tomas Liptak • Aladar Madari • Ivana Grulova • Lucia Slovinska • Miriam Nagyova • Dasa Cizkova

Received: 28 January 2014 / Accepted: 15 May 2014 Ó Springer-Verlag Italia 2014

Abstract Inosine, a purine nucleoside, is one of the novel substances, which can preserve the neuronal and glial viability and stimulate intact neurons to extend axons. We, herein, evaluated the effect of oral inosine treatment on spinal cord injury (SCI) recovery by means of locomotor and bladder function, quantification of neurons and spinal cord tissue sparing. Rats after compression SCI were divided into groups—SCI-Aqua and SCI-Inosine (daily application of aqua for injection or inosine)—locomotion of hind limbs (BBB score) and urinary bladder function were evaluated from day 1 to 28 after SCI. The neuronal profile was determined by immunohistochemistry with NeuN antibodies and tissue sparing by Luxol fast blue staining method. SCI affected the functional movement of hind limbs in both groups with gradual improvement (increased BBB score) during survival. However, we found a significant difference in BBB score and recovery of bladder function between SCI-Aqua and SCI-Inosine groups during the second week of survival following SCI. In addition, the number of NeuN positive cells and percentage of tissue sparing was also significantly higher in SCI-Inosine group when compared with the SCI-Aqua group. Daily oral administration of inosine after SCI throughout the survival was beneficial for locomotion and micturition, neuronal survival and tissue sparing. This indicates that inosine may represent one of the coM. Kuricova (&)  V. Ledecky  T. Liptak  A. Madari Small Animal Clinic, University of Veterinary Medicine and Pharmacy, Komenskeho 73, 041 81 Kosice, Slovakia e-mail: [email protected] I. Grulova  L. Slovinska  M. Nagyova  D. Cizkova Institute of Neurobiology, Slovak Academy of Sciences, Centre of Excellence for Brain Research, Soltesovej 4-6, 040 01 Kosice, Slovakia

stimulatory factors for treatment strategies to promote neuronal plasticity after SCI. Keywords Spinal cord injury  Inosine  Urinary bladder  Functional recovery

Introduction Spinal cord injury (SCI) is often associated with an unfavorable prognosis for recovery and hence, quality of life and the outcome after SCI are still unpredictable. The extent of damage, depending on the cause of trauma, is directly linked to the prognosis [1–4]. Initial mechanical disruption of the spinal cord (SC) structures is followed by the cascade of secondary events—the main cause of progressive cell damage within the grey matter (GM) progressing to the white matter (WM) [5]. The most common treatment for acute SCI is early stabilization followed by decompression and minimizing the secondary changes [6]. Cells of the central nervous system (CNS) and their axons in adult mammals have limited ability to regenerate and make functional synapses after injury which has been ascribed to inhibitory proteins in myelin [7–9] and lack of essential trophic factors [10, 11]. Inosine is often used as a dietary supplement which can reduce the spread of secondary injury after SCI [12–15]. Although the specific mechanisms of inosine action are still not fully understood, in some neurons, it regulates the expression of genes involved in axon outgrowth [16]. Axonal rewiring was confirmed in rats [17, 18] and inosine-induced neuroprotective effect against hypoxia/ischemia was demonstrated in vitro [19, 20] and in vivo [21]. The aim was to evaluate the efficacy of orally administered inosine on SCI recovery by means of motor

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function (MF) of hind limbs [22], urinary bladder function (UBF), SC tissue sparing of GM and WM and neuronal profile after compressive SCI in rats. Present data demonstrate beneficial inosine effect on all examined parameters, which more likely represent complementary approach therapy to enhance neuronal plasticity.

Materials and methods The SCI was performed in 16 adult male Wistar rats (body weight 290–310 g). The animals were anesthetized (2–3 % isoflurane induction-box) and anesthesia was maintained via inhalation mask (1–2 % isoflurane). A laminectomy was performed on the Th10 vertebra and modified 2-French Fogarty catheter was inserted into the epidural space [23]. Balloon of this catheter was inflated with saline (12.5 ll) for 5 min. After deflation and removal, closure of the wound and disconnection from anesthesia were performed. The animals were recovered in warmed cages, observed for 120 min after SCI, and BBB score = 0 for complete paralysis of hind limbs was recorded. The rats were randomly divided into groups: (a) SCIAqua application of aqua for injection (n = 8) and (b) SCIInosine-application of inosine (n = 8), started 2 h after SCI. In the SCI-Sham group (n = 8), the catheter was inserted at the same level, but balloon was not inflated and no lesion was caused. Rats in each group survived for 14 (14D) or 28 (28D) days. Until the autonomous UBF regained, all animals required manual emptying 2–3x/day after SCI. Since inosine is available as an oral supplement, we decided for its direct administration per os. We used a thick walled rubber tube attached to a syringe to deliver the compound directly into the stomach. The length of the tube was determined by measuring the distance from rat’s mouth to the last rib. All animals were used to be restrained in vertical position, with extension of the head, which allowed the tube to smoothly pass down into the stomach. The SCI-Aqua group received 4 ml aqua for injection and the SCI-Inosine group received inosine (400 mg capsule) dissolved in 2 ml aqua for injection ?2 ml to flush the residual inosine, daily throughout survival. During survival, the rats were kept under standard conditions (temperature of 22–24 °C, natural light cycle), while being fed and watered ad libitum. This study was performed with the approval and according to the guidelines of the Institutional Animal Care and Use Committee of the Slovak Academy of Sciences and with the European Communities Council Directive (2010/63/EU) regarding the use of animals in research, Slovak law for animal protection No. 377/2012 and 436/2012. All rats were evaluated before SCI, 2 h after SCI, and at 1, 4, 7, 10, 14, 17, 21, 24, and 28D after SCI. We analyzed

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the MF according to Basso–Beattie–Bresnahan score [22] which represents 21 points, where 0 reflects no MF and 21 reflects normal MF. Each rat was tested for 5 min; rat’s hind limb movements, trunk position, stability, stepping, coordination, paw placement, toe clearance, and tail position were analyzed. All rats underwent manual bladder expression for various time after SCI. We established the four-point urinary bladder function score (UBFS) which was evaluated daily. 0 = complete loss of function (manual expression of bladder 39/day, the urine contains blood), 1 = partial recovery of UBF (manual expression 29/day, urine may contain blood), 2 = partial recovery of UBF (partial/ complete release of sphincter spasm, manual expression 19/day, still blood in urine), 3 = advanced recovery (manual expression 1x/day, no blood in urine), 4 = physiological UBF. Experimental rats were euthanized at 14D (n = 8) or 28D (n = 8) after SCI by inducing deep general anesthesia (intraperitoneal thiopental injection-50 mg/kg). All animals were perfused transcardially with 500 ml saline, followed by 500 ml of 4 %paraformaldehyde in 0.1 M phosphate-buffered saline. Each SC was dissected into 1.5 cm blocks (L: central lesion segment, R: rostral segment, and C: caudal segment). 40 lm thick transverse cryostat (leica instruments, Heidelberg, Germany) sections were cut serially and representative sections were collected for immunohistochemical labeling (15 sections/animal) and for Luxol fast blue (LFB) staining (15 sections/animal). Sections were incubated in primary antibody: mouse anti-NeuN (1:500, Millipore, clone A60, MAB377) for 24 h at 4 °C. Afterward, free-floating sections were washed with 0.1 M PBS and incubated at room temperature for 2 h with secondary fluorescent antibody: anti-mouse, conjugated with Texas red (Alexa flour 594, molecular probes). Sections were washed with 0.1 M PBS, mounted, and coverslipped with Vectashield medium (Vector Laboratories. Sections for modified LFB labeling were incubated with LBF (10 min), rinsed 3–59 in 95 % alcohol and washed in distilled water (DW). Differentiation was performed in lithium carbonate solution (2 min) with following double dipping in 95 % alcohol and rinsing in DW. Afterwards, sections were counter stained by cresyl violet for 1 min and washed in DW. Differentiation continued by dehydration in 95 % alcohol (3–5 min) followed by clearing in xylene (3 min). Sections were coverslipped with mounting medium (Enthelan). Sections were analyzed using Nicon-Ti inverted fluorescence-microscope w/Perfect Focus (LHRRB 113) at 49 and 109 magnification and captured with digital camera iXon DU897EMCCD. The number of NeuN-positive cells was evaluated through a sample field (250 9 250 lm) bilaterally in GM. Analysis of spared tissue was performed by outlining the GM and

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WM, digitized, and calculated with ImageJ software, while two-dimensional area expressed by pixels was calculated as percentage of spared tissue within 1 mm2. The differences between all evaluated groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hock tests. All the data are presented as mean values ± SD. Differences were considered statistically significant **P \ 0.01, *P \ 0.05.

Results The SCI caused complete paralysis in all rats 24 h post surgically. The gradual recovery was observed in both experimental groups (Fig. 1). Significant MF improvement was detected in the SCI-Inosine group when compared with SCI-Aqua at 7, 10 and 14D after SCI (*P \ 0.05). BBB score in the SCI-Inosine group reached maximum at the end of survival interval (28D/15.88 ± 1.31) in comparison to the SCI-Aqua (28D/11.25 ± 1.55). The SCI-Sham group showed normal MF associated with BBB score 21 throughout the survival. The experimental groups required manual bladder emptying 2–39/day during first week. The urine of most animals contained blood which disappeared with time. In the SCI-Inosine group, we observed a significant improvement and only occasional blood in urine at 7D (**P \ 0.01) and 10D (*P \ 0.05) after SCI. Full recovery of BF was manifested in the SCI-Inosine group earlier (UBFS:14D/4.0), which is in contrast to the SCI-Aqua group (UBFS:14D/3.25, 21D/3.75, 24D/4.0) (Fig. 2). The immunohistochemistry with NeuN-antibody (Fig. 3) was used for counting neuronal profiles. An increased number of NeuN-positive neurons in entire GM was detected over time in both groups. The number was significantly higher in the SCI-Inosine group, compared with the SCI-Aqua (14D: SCI-Aqua = 238.1 ± 19.91, SCI-Inosine = 341.9 ± 31.54; 28D: SCI-Aqua = 317.3 ± 24.18, SCI-Inosine = 404.9 ± 37.67). Statistical differences were detected at both survival periods (**P \ 0.01, *P \ 0.05) (Fig. 4). Except for a significantly higher number of neurons we observed pronounced axonal ramification in the SCI-Inosine group (Fig. 3b, e). These facts are associated with better MF and higher BBB score as well as with earlier UBF recovery. We evaluated the amount of spared GM and WM in 3 spinal segments at 14D and 28D after SCI. Inosine significantly reduced the tissue losses at 14D in WM in the rostral segment (R: 14D/WM-Aqua 46.96 ± 11.19 %; WM-Inosine 57.64 ± 12.23 %) and in GM in the caudal segment (C: 14D/GM-Aqua 15.46 ± 1.14 %; GM-Inosine 19.42 ± 1.92 % (*P \ 0.05). At 28D after SCI there was evident statistical difference in tissue sparing in the lesion

Fig. 1 Evaluation of motor function of hind limbs using BBB score in the open field in rats after experimental spinal cord injury, performed at 1, 4, 7, 10, 14, 17, 21, 24 and 28 days after SCI. The significant difference in BBB score between the SCI-Aqua and the SCI-Inosine group was determined during the second week of survival (one way ANOVA followed by Tukey’s post-hoc tests, data are expressed as mean ± SD. Statistical difference *P \ 0.05)

Fig. 2 Improvements of the urinary bladder function after SCI. We established the four-point urinary bladder function score (UBFS). Significant differences in recovery of bladder function between experimental groups SCI-Aqua and SCI-Inosine was recorded for day 7 and 10 after injury, followed by non-significant gradual improvement (one way ANOVA followed by Tukey’s post-hoc tests, data are expressed as mean ± SD. Statistical difference **P \ 0.01, *P \ 0.05

segment (L: 28D/WM-Aqua 45.05 ± 9.39 %; WM-Inosine 54.56 ± 12.22 %; *P \ 0.05) and in the caudal segment (C: 28/WM-Aqua 47.84 ± 6.46 %; WM-Inosine 64.22 ± 3.49 %; **P \ 0.01) (Fig. 5).

Discussion Inosine has been often used by athletes to handle strenuous exercise or intense training without muscle fatigue. A normal dose of inosine (50 mg/kg of body weight/day) is able to increase the metabolic activity that boosts ATP production for respiration and oxygen transport. Inosine has broad anti-inflammatory effects including inhibition of

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Fig. 3 Representative transversal spinal cord sections from the rostral segment (Th7) with NeuN-positive neurons in experimental groups SCI-Aqua (a–c) and SCI-Inosine (d–f) at day 28 after SCI. Except for a significantly higher number of neurons, we also observed

pronounced axonal ramification in SCI-Inosine group. These facts are associated with better locomotor function and higher BBB score in SCI-Inosine group as well as with earlier urinary bladder function recovery. Scale bar: a, d 200 lm; b, e 100 lm; c, f 50 lm

pro-inflammatory substances and protection from endotoxin-induced inflammation and reperfusion injury [24]. Furthermore, inosine is a precursor of uric acid which is a potent antioxidant and it is believed to block the effect of toxic free-radicals. This effect is presently being tested in studies and clinical trials for neurological diseases [25–29]. The most common complications of SCI are disorders of UBF often accompanied by inflammation. It has been shown that SCI-induced bladder dysfunction resulting in hematuria and cystitis can be eliminated by daily oral administration of inosine. Furthermore, inosine treated rats showed significantly better contractile responses of urinary bladder [30, 34]. Our data correlate with these findings and confirm that SCI-Inosine treated rats (19/daily/400 mg/ animal) recovered faster, they began to urinate normally already within the first week after injury. This is most likely due to inosine mediated rewiring of lost bladder

fibers with functionally relevant synaptic re-formations. Because, inosine is a naturally occurring derivative of adenosine, we can speculate that it may have a similar protective and anti-inflammatory effect on bladder as adenosine in the heart and other organs [35]. Furthermore, all four adenosine receptors A1, A2A, A2B and A3 receptors are expressed in the uroepithelium, where adenosine is produced [36]. Thus, the biological actions of inosine might involve effects on adenosine receptors through A1 receptor, while the immunomodulatory effect is most likely mediated through A3 or A2A receptors [37]. Previous in vitro studies confirmed that inosine may play a dual role. Stimulates mast-cell degranulation resulting to pro-inflammatory effects, but on the other hand it is able to suppress macrophages and lymphocytes and reduce the production of pro-inflammatory cytokines (TNF-a, IL-12, MIP-a IFN-c) [38, 39]. Finally, systematic administration

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Fig. 4 Quantification of spinal NeuN-positive neurons in the rostral segment in rats 14 and 28 days after SCI. Immunohistochemistry with NeuN antibodies was used for counting neuronal profile present in the dorsal and ventral spinal cord horns. In both groups, SCI-Aqua and SCI-Inosine, an increased number of NeuN positive neurons in entire grey matter (GM) layers was detected over time. The number of NeuN positive neurons was significantly higher in inosine treated animals and the differences between groups SCI-Aqua and SCIInosine were detected at both survival periods (one way ANOVA followed by Tukey’s post-hock tests, data are expressed as mean ± SD. Statistical difference **P \ 0.01; *P \ 0.05)

of inosine in mice ameliorated the pro-inflammatory cytokine ‘storm’ following endotoxin administration, which usually leads to death by injuring multiple organs [40]. In line with these findings is the fact that inosine also attenuates the course of various chronic autoimmune diseases or ischemia–reperfusion injury by decreasing immune activation [41, 42]. However, how inosine regulates the physiological and pathophysiological processes in the bladder is less well understood than in immune and nervous systems [33, 37, 43]. Recent studies confirmed that inosine induces significant axonal reorganization [32, 33] which is linked to the fact that inosine can enhance the MF recovery after CNS injury [18, 33]. In our study, we observed a significant improvement in motor function during the second week after SCI. The maximum BBB score at the end of survival did not show significant difference between groups, suggesting that inosine did not affect the extent of recovery, but only the speed of functional improvement. Similar considerations are valid also for the urinary bladder function. Thus, it is likely that the therapeutic effects of inosine are mediated by the suppression of the initial inflammatory responses. The reason why inosine is effective predominantly during first 2 weeks after SCI is most likely attributed to its anti-inflammatory and immunomodulatory effect which influences the initial

pathophysiological processes. We could hypothesize that fixed dose of inosine could result in the tolerance and be ineffective during longer survival. Therefore, different dose regiments should be tested in further studies. Yoles et al. [44] reported significant reduction of the number of neurons after SCI associated with alteration of MF. This is most likely attributed to delayed degeneration of initially spared neurons. We have partially confirmed this fact in the present study by quantitative immunohistochemical analysis documenting a significantly higher number of NeuN-positive cells in SC sections from the SCI-Inosine group, when compared to SCI-Aqua group. In addition to a significantly higher number of neurons, there was also noticeable delicate ramification of these neurons. On the contrary, the SCI-Aqua group sections contained only few NeuN-positive motor neurons revealing almost no or only poor ramification, which might correlate with early degenerative processes. These neuroplasticity changes after SCI can be mediated in part by compensatory circuits of the corticospinal tract (CST) and other major pathways. Inosine administration enables the undamaged or severed CST axons to extend collateral branches into the denervated side of the spinal cord. This mechanism is partially mediated through inosine-induced neuronal Mst3b activation, as a part of a signal transduction pathway that regulates axon growth [45]. Although it is possible to deliver inosine intraperitoneally [30, 31] or by cortical/cisternal infusion [32, 33], we have chosen oral gavage to deliver the constant dose. This method is safe and easy to use, without the need of other equipment or anesthesia. Thus, in the present study, we noted very good outcomes in all measured endpoints with no side effects during survival. Benowitz et al. [32] in their studies on rats used a dose of inosine about 1000 times higher than the estimated effective dose. We used approximately ten times lower dose (1.2 g/kg) to ensure maximum support for SC tissue knowing that inosine can ameliorate plasticity and cell loss, antioxidant and antiinflammatory activity, immunomodulation, neuroprotection and axon collateral growth, and thus functionality enhancements. Because of the specific pharmacokinetics and pharmacodynamics, we choose a single high dose per day in order to maximize the positive therapeutic effect. However, it will be necessary to determine the concentration of inosine in blood, together with possible multiple doses per day. Furthermore, SCI in our study caused a widespread loss of gray and white matter within all evaluated segments with more profound loss of tissue in the SCI-Aqua group. Here we also assume that the effect of inosine on the overgrowth and the formation of collaterals after CNS injury may particularly enhance the sparing of the WM tissue.

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Fig. 5 Percentage of tissue sparing on transversal sections of spinal cord measured in grey (GM) and white (WM) matter. Inosine significantly reduced the total volume of spinal cord degenerative areas at day 14 in WM in the rostral segment (R: 14D/WM-Aqua 46.9 ± 11.19 %; WM-Inosine 57.64 ± 12.23 %) and in GM in the caudal segment (C: 14D/GM-Aqua 15.46 ± 1.14 %; GM-Inosine

19.42 ± 1.92 %). At day 28 there was evident statistical difference in tissue sparing between experimental groups in WM in lesion site (L: 28D/WM-Aqua 45.05 ± 9.39 %; WM-Inosine 54.56 ± 12.22 %) and in caudal segment (C: 28/D WM-Aqua 47.84 ± 6.46 %; WMInosine 64.22 ± 3.49 %). Data are expressed as mean ± SD. Statistical difference **P \ 0.01; *P \ 0.05

Our study concludes that it is necessary to study inosine as a powerful therapeutic supplement for damaged nervous tissue. Thanks to its very low peroral toxicity [46] and large therapeutic range it is preferable to administer inosine orally in humans and in veterinary practice. Significant effects of inosine after SCI remain to be verified during a longer administration period, e.g. 3–6 months. In those treatments, it would be possible to identify potential side effects due to its long-term use even though a low toxicity has been reported.

after inosine administration. This indicates that inosine may represent one of the therapeutic strategies to promote regeneration and functional recovery after spinal cord injury in clinical practice.

Conclusion The purine nucleoside inosine, given to rats orally in dose 1.2 g/kg 2 h after experimental spinal cord injury and then continuously daily for 28D, positively affected the speed of locomotor function of hind limbs and urinary bladder function recovery. Furthermore, the number of neurons was higher and more of gray and white matter tissue was spared

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Acknowledgments This work was supported by the financial support from the Scientific Grant Agency of Ministry of Education, Science, Research and Sport of the Slovak Republic and the Slovak academy of Sciences (VEGA no. 1/2945/12, VEGA no. 2/0169/13) and the Slovak Research and Development Agency (APVV no. 0472/11).

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Oral administration of inosine promotes recovery after experimental spinal cord injury in rat.

Inosine, a purine nucleoside, is one of the novel substances, which can preserve the neuronal and glial viability and stimulate intact neurons to exte...
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