l -Theanine improves functional recovery after traumatic spinal cord injury in rats

l -Theanine improves functional recovery after traumatic spinal cord injury in rats

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Journal of the Formosan Medical Association xxx (xxxx) xxx

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.jfma-online.com

Original Article

L-Theanine

improves functional recovery after traumatic spinal cord injury in rats Chih-Chuan Yang a,b,1, Kuo-Chi Chang c,1, Mao-Hsien Wang d, Hsiang-Chien Tseng e,f, Hung-Sheng Soung g,h, Chih-Hsiang Fang i, Yi-Wen Lin i, Keng-Yuan Li i, Cheng-Chia Tsai a,b,* a

Department of Neurosurgery, Mackay Memorial Hospital, Taipei, 10449, Taiwan, ROC Department of Medicine, Mackay Medical College, New Taipei City, 252, Taiwan, ROC c Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, 10608, Taiwan, ROC d Department of Anesthesia, En Chu Kon Hospital, Sanshia District, New Taipei City, 23702, Taiwan, ROC e Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, 11101, Taiwan, ROC f School of Medicine, Fu Jen Catholic University, New Taipei City, 24205, Taiwan, ROC g Department of Psychiatry, Yuan-Shan Br. of Taipei Veteran General Hospital, Yilan County, 26604, Taiwan, ROC h Department of Biomedical Engineering, National Defense Medical Center, Taipei, 11490, Taiwan, ROC i Institute of Biomedical Engineering, National Taiwan University, Taipei, 10051, Taiwan, ROC b

Received 5 July 2019; received in revised form 21 August 2019; accepted 13 November 2019

KEYWORDS Apoptosis; Inflammation; L-theanine; Oxidative stress; Spinal cord injury

Background/Purpose: Spinal cord injury (SCI) is a devastating medical condition for which no effective pharmacological interventions exist. L-Theanine (LT), a major amino acid component of green tea, exhibits potent antioxidative and anti-inflammatory activities and protects against various neural injuries. Here, we evaluated the potential therapeutic effects of LT on the recovery of behavioral motor functions after SCI in rats and the underlying neuroprotective mechanisms. Methods: SCI was induced by applying vascular clips to the dura through a four-level T5eT8 laminectomy, and saline or LT (10/30 mg/kg) was intrathecally administered at 1-, 6-, and 24-h post-SCI. At 72-h post-SCI, half of the rats from each group for each parameter were sacrificed, and their spinal cord was excised for measurement of malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase, catalase, tumor necrosis factor-a, interleukin-1b/-6, myeloperoxidase, and caspase-3. The remaining rats from each group were subjected to Bresnahan

* Corresponding author. Department of Neurosurgery, Mackay Memorial Hospital, No. 92, Sec. 2, Zhongshan N. Rd., Taipei City, 10449, Taiwan, ROC. Fax: þ886 2 2543 3642. E-mail address: [email protected] (C.-C. Tsai). 1 The two first authors contributed equally to this work. https://doi.org/10.1016/j.jfma.2019.11.009 0929-6646/Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009

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C.-C. Yang et al. locomotor-rating scale (BBB), inclined-plane, toe-spread, and hindfoot bar-grab tests at 1-, 4-, 7-, 10-, and 14-days post-SCI. Results: LT treatment reduced NO and MDA levels, increased antioxidative strength, and markedly suppressed the levels of neuroinflammatory and apoptotic markers in the spinal cord after SCI. Moreover, LT treatment drastically promoted the recovery of behavioral motor functions post-SCI. Conclusion: Our findings revealed that LT can enhance the recovery of behavioral motor functions after SCI in rats, which related to the suppression of post-traumatic oxidative response, neural inflammation, and apoptosis. This evidence indicates that LT holds considerable potential for use in the clinical treatment/prevention of SCI-induced motor dysfunction. Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Introduction Spinal cord injury (SCI), one of the most devastating medical conditions, can impair physical functions temporarily or permanently. SCI is characterized by initial physical damage (primary injury) leading to one or more continuous damaging processes (secondary injury) that spread away from the injury epicenter and cause motor, sensory, and/or autonomic dysfunction.1 Although the primary injury causes tissue necrosis and disruption of neuronal and vascular structures, secondary injury is predominantly responsible for SCI-associated damage, including oxidative stress, inflammation, necrosis, ischemia, neuronal apoptosis, and, ultimately, glial scar formation.2e5 Although several therapeutic methods have been developed to improve outcomes in SCI patients, including neural stem cell transplantation, tissue engineering, and molecular therapy, post-SCI neurological recovery remains limited. However, numerous aggravating factors associated with the secondary injury could be considered to represent targets for therapeutic intervention,1,6 and previous studies suggest that antioxidant or anti-inflammatory agents might improve SCI treatment in animal models.2e5,7,8 L-Theanine (LT), a chemical structurally analogous to glutamate and glutamine, constitutes 0.5%e2% of the dry weight of tea and is synthesized from ethylamine and glutamate in tea leaves. LT is the main non-protein amino acid component responsible for greenetea flavor and taste and thus determines the quality of green tea. LT is rapidly absorbed into blood from the intestinal tract through cotransport with Naþ and then further redistributed to other organs, including the brain.9 LT reportedly does not produce toxic effects in animals or humans, and because of its tasteenhancing property and promising health benefits, it has been approved as a generally regarded as safe ingredient by the United States Food and Drug Administration and is widely used in food and pharmaceutical industries.9,10 LT exerts numerous pharmacological effects, such as sedative, hypotensive, anti-obesity, and anti-inflammatory effects,10e13,35 can scavenge reactive radicals and alleviate peroxidative conditions, and is expected to exhibit neuroprotective properties suitable for treating behavioral impairments induced by toxins and stress.11,14e17 However, reports on LTrelated protective effects on post-SCI behavioral motor

dysfunction remain limited. Given its antioxidative and antiinflammatory properties, we hypothesized that LT can counteract SCI-increased neural damage and improve recovery of behavioral motor functions post-SCI. Here, we examined the potential therapeutic effects of LT on oxidative stress, antioxidation strength, neuroinflammation, and apoptosis in spinal cord tissue and on motor-function recovery post-SCI. Malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, IL-6, myeloperoxidase (MPO), and caspase-3 were measured in control, sham, and SCI groups treated with/without LT, and neuronal behavioral motor functions were assessed using Basso Beattle Bresnahan locomotor-rating scale (BBB), inclined-plane, toe-spread, and hindfoot bar-grab tests.

Materials and methods Ethical approval All animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University College of Medicine and College of Public Health (IACUC Approval No: 20170025).

Drug LT (N-ethyl-L-glutamine, 98%; SigmaeAldrich, St. Louis, MO, USA) was prepared in physiological saline solution with distilled water. Drug doses were adapted from previous reports.11,14

Animals Male Wistar rats (300e320 g, w3-months old) were housed in Plexiglas cages (3 rats per cage) with free access to food and water in a room under controlled temperature (22  3  C) and a 12/12-h light/dark cycle (lights on: 7:00 A.M.). Considerable effort was devoted to minimizing the number of animals used and their suffering.

Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009

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neuroprotection in spinal cord injury

Animals were randomly allocated to the following groups (N Z 14/group) and subjected to SCI or laminectomy only and then intrathecally administered saline or LT (10/30 mg/ kg) within the first hour and at 6- and 24-h post-surgery: SCI þ saline (SCI) SCI þ LT 10 mg/kg (SCI þ T10) SCI þ LT 30 mg/kg (SCI þ T30) Sham þ saline (Sham; same surgical procedures as the SCI groups, except that aneurysm clip was not applied) 5. Sham þ LT 10 mg/kg (Sham þ T10) 6. Sham þ LT 30 mg/kg (Sham þ T30) 7. Control (C): no surgical or medical intervention (except anesthesia)

1. 2. 3. 4.

At 72-h post-surgery, half of the rats from each group for each parameter were sacrificed, and their spinal cord was excised for biochemical studies. The remaining rats were subjected to behavioral assessment at 1-, 4-, 7-, 10-, and 14-days post-surgery. During this study, six rats died naturally for unknown reasons.

SCI Animals were anaesthetized using chloral hydrate (400 mg/ kg body weight). A longitudinal incision was made on the midline of the back to expose the paravertebral muscles, which were dissected away to expose T5eT8 vertebrae. The spinal cord was exposed through a four-level T5eT8 laminectomy, and SCI was produced through extradural compression of the spinal cord using an aneurysm clip (closing force: 55 g).3 In all injury groups, the spinal cord was compressed for 1 min. Sham-group animals were subjected to laminectomy only. After surgery, 1 ml of saline was administered subcutaneously to replace the blood volume lost during the surgery. During recovery from anesthesia, the rats were placed on a warm heating pad and covered with a warm towel. Food and water were provided ad libitum. During this period, animal bladders were manually voided twice daily until the rats regained normal bladder function.

Sample preparation and biochemical experiments At 72-h post-SCI, animals were sacrificed using a CO2 chamber (to minimize stress). A 2-cm segment of spinal cord containing the injury area was dissected, washed with ice-cold phosphate-buffered saline (PBS), snap-frozen in liquid nitrogen, and stored at 80  C. For biochemical measurements, samples were homogenized in ice-cold PBS containing 1% (v/v) protease-inhibitor mixture (SigmaeAldrich), lysates were centrifuged (15,000g, 20 min, 4  C), and the supernatants and pellets were collected and stored at 80  C until analyses.

Measurement of oxidativeestress parameters MDA To determine tissue lipid peroxidation, MDA concentration was measured based on MDA reaction with thiobarbituric acid (TBA) (Esterbauer and Cheeseman, 1990). Briefly,

3 150-ml supernatant samples were each mixed with 300 ml of trichloroacetic acid (20%; SigmaeAldrich) and TBA (0.67%; SigmaeAldrich), heated in boiling water for 60 min, cooled at room temperature, and centrifuged (3500g, 10 min). The 532-nm absorbance of the samples was measured, and tetramethoxypropane (SigmaeAldrich) was used to prepare the standard curve. MDA concentrations were reported as nmol/mg protein. Nitrite To measure nitrites (NOs) as oxidants in spinal cord samples, 150 ml of supernatant was mixed with sodium hydroxide (0.3 M), and 5 min later, 75 ml of zinc sulphate (5%) was added to precipitate proteins, followed by centrifugation of the mixture (15,000g, 20 min, 4  C) and separation of the supernatant. To 200 ml of the supernatant, 300 ml of vanadium (III) chloride (80 mg of VCl3 in 10 ml of 1 M HCl; SigmaeAldrich) was added, and the mixture was incubated at 37  C for 45 min, followed by measurement of the absorbance at 540 nm. Sodium nitrite was used to plot the standard curve,18 and results are presented as nmol/mg protein. SOD SOD activity was determined, as described previously.19 The method is based on the inhibition of nitro blue tetrazolium reduction by the xanthineexanthine oxidase system acting as a superoxide generator. SOD activity was assessed in the ethanol phase of the supernatant after 1 ml of ethanol/chloroform mixture (5/3, v/v) was added to 1 ml of each sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition of nitro blue tetrazolium reduction. SOD activity is expressed as U/mg protein. CAT CAT activity was assayed based on the rate of decrease of hydrogen peroxide (H2O2) absorbance at 240 nm.20 H2O2 decomposition at 25  C (in mmol H2O2 per min), which produces the lysate by catalyzation, was used to define catalase activity. Total protein concentration in cell lysates was determined using bovine serum albumin as the standard.21 Results are expressed as U/mg protein.

Measurement of neuroinflammatory markers TNF-a and IL-1b/-6 To investigate inflammation levels, the proinflammatory factors TNF-a, IL-1b, and IL-6 were measured in spinal cord tissues using commercial ELISA kits (Abcam, Cambridge, UK) according to manufacturer instructions. All measurements were performed in duplicate, and results are presented as pg/mg protein. MPO MPO activity was measured as an indicator of neutrophil accumulation in spinal cord samples. Tissue pellets were resuspended in ice-cold phosphate buffer (50 mM; pH 6) containing 0.5% hexadecyltrimethylammonium bromide (SigmaeAldrich), and the homogenates were frozen in liquid nitrogen and thawed in three consecutive cycles,

Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009

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4 with sonication (10 s, 4  C) between cycles. After the final sonication, samples were centrifuged (15,000g, 20 min, 4  C), and the supernatant was used for MPO assays. The rate at which a colored product formed during MPOdependent reaction with o-dianisidine (0.167 mg/ml; SigmaeAldrich) and H2O2 (0.003%) was measured at 460 nm. One unit of MPO was defined as the amount that degrades 1 mmol of H2O2 per minute at 25  C.22 Results are expressed as mU/mg protein.

Apoptosis marker Caspase-3, an executioner molecule in apoptotic cascades, was measured based on the cleavage of the chromogenic caspase substrate Ac-DEVD-pNA. Caspase-3 is a protease that is rapidly activated when cells are exposed to apoptotic conditions and then cleaves poly(ADP-ribose) polymerase. Caspase-3 amount was determined through spectrophotometric measurement (at 405 nm) according to manufacturer instructions (R&D Systems, Minneapolis, MN, USA).

Behavioral assessment Post-SCI behavioral recovery of hindlimbs from motor disturbances was assessed using the BBB test,23 inclined-plane test,24 and toe-spread and hindfoot bar-grab tests.24 All tests were conducted in a quiet, isolated room in the animal facility unit at National Taiwan University College of Medicine and College of Public Health. To eliminate experimenter subjective bias, each animal was arbitrarily assigned a number, and all functional and behavioral tests and clinical-assessment evaluations were performed independently by two experienced co-workers who were blinded to all treatments in all experiments. The scores of each test were evaluated at 1-, 4-, 7-, 10-, and 14-days post-SCI for each animal in the same sequence, with a 20-min rest between each test. At each test session, three separate measurements were obtained for each animal, with the mean value being considered a single score for the animal. BBB test A rat was placed in an open-field box (150-cm long, 120-cm wide, 30-cm high) with a smooth floor, and two independent examiners studied its locomotor ability. Rats were tested in pairs for 4 min. During open-field testing, rats were encouraged to locomote continuously. All hindlimb movements were recorded, except for those that were part of a reflex or elicited by contact with an examiner or another animal. The BBB test describes hindlimb joint movement, hindpaw placement, and forelimb-hindlimb coordination according to a scale from 0 (complete paralysis) to 21 (complete mobility).23 Inclined-plane test The test involved measuring the maximum angle at which an animal could support its body weight for at least 5 s on an inclined board. Rats were placed facing downwards on an inclined board covered with a rubber mat, and the board was raised in 5 increments during the process. The

C.-C. Yang et al. maximum angle (0 e80 ) at which the rat was able to maintain its position was recorded(Rivlin and Tator, 1977). Toe-spread and hindfoot bar-grab tests Post-SCI functional deficits were tested by scoring toe spread and hindfoot bar grab, as described previously.24 Toe spread is a reflex elicited by lifting a rat. The rat is placed on a table and then held by the body with one hand and lifted, with its legs allowed to hang free. The amount of toe spreading in each hindlimb is graded individually, as follows: 0, no spreading; 1, minimal spreading with flaccid toes; 2, partial spreading; and 3, regular complete toe spread. Hindfoot bar grab is a reflex elicited by hindfoot contact with a small-diameter bar and graded, as follows: 0, no response when hindfoot touches bar; 1, hindfoot responds to contact with bar but cannot grab the bar; 2, hindfoot grabs bar but only weakly; and 3, hindfoot successfully grabs bar and pulls it against the body.

Intrathecal injection Animals were administered intrathecal lumbar injections of LT (10/30 mg/kg) at L3eL4 of the spinal column to avoid damaging the spinal cord. The pH of each LT solution was set to between 7.2 and 7.4 to match the cerebrospinal fluid pH. Animals were anesthetized through 4% isoflurane inhalation, and then 50 ml of saline or LT was injected over a 1-min period into the intrathecal space, with 50 ml selected to ensure that the solution diffused to cervical segments. The animals were alert and mobile moments after the inhalation anesthesia was removed and displayed no signs of distress or neural damage.

Statistical analysis Data from behavioral studies are presented as the mean  standard error of the mean (SEM). Data were analyzed using one-way analysis of variance (ANOVA), followed by a Tukey pairwise post hoc test. A P < 0.05 was considered statistically significant.

Results LT inhibited post-SCI increase in spinal cord MDA and NO production LT effects on MDA and NO levels in the spinal cord after SCI were measured (Fig. 1). At 72-h post-SCI, MDA and NO were significantly increased in the SCI group (t test; C vs. SCI; P < 0.001), from 0.959 nmol/mg protein to 4.033 nmol/mg protein and 0.597 nmol/mg protein to 2.653 nmol/mg protein, respectively. Post hoc analysis revealed that LT exerted no effect on MDA and NO in Sham groups but dosedependently inhibited the increase in MDA and NO levels in SCI groups (P < 0.001): 10 mg/kg and 30 mg/kg LT, respectively, reduced MDA and NO by 22.99% and 24.35% (4.033e3.106 nmol/mg protein and 2.653 to 2.007 nmol/mg protein, respectively) and 48.85% and 50.66% (4.033e2.063 nmol/mg protein and 2.653 to 1.309 nmol/mg protein, respectively). These results suggest that LT

Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009

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Figure 1 LT treatment inhibits increase of spinal cord MDA and NO production after SCI. LT effects on MDA and NO levels in rat spinal cord post-SCI were measured. Data represent the mean  SEM (N Z 7). Treatment groups: SCI: SCI þ saline; SCI þ T10: SCI þ LT 10 mg/kg; SCI þ T30: SCI þ LT 30 mg/kg; Sham: laminectomy þ saline; Sham þ T10: laminectomy þ LT 10 mg/kg; Sham þ T30: laminectomy þ LT 30 mg/kg; C: control. ***P < 0.001 vs. C, ###P < 0.001, ##P < 0 01 vs. SCI; data were analyzed by mixed repeated ANOVA and post hoc using one-way ANOVA with Tukey pairwise tests.

alleviated the oxidative and nitrosative stresses that were increased in rat spinal cord after SCI.

enhanced the antioxidative strength that was decreased post-SCI in rat spinal cord.

LT prevents post-SCI reduction in spinal cord antioxidative strength

LT reduced post-SCI increases in spinal cord neuroinflammatory factor levels and neutrophil accumulation

We then assessed how LT affects the protective enzymes SOD and CAT in the spinal cord after SCI (Fig. 2). SOD and CAT levels were decreased in the SCI group at 72-h post-SCI by 74.63% and 54.70%, respectively (from 1.363 to 0.346 nmol/mg protein and 27.943 to 12.657 nmol/mg protein; t test, C vs. SCI; P < 0.001). Post hoc analysis results showed that although SOD and CAT in Sham groups were unaffected by LT, the diminished SOD and CAT levels in SCI groups were restored in a dose-dependent manner by 10 mg/kg and 30 mg/kg LT. These findings suggest that LT

We then quantified LT effects on TNF-a, IL-1b, IL-6, and MPO levels in the spinal cord after SCI (Fig. 3). All four factors were significantly increased at 72 h in the SCI group (t-test, C vs. SCI; P < 0.001 in all cases), as follows (in nmol/mg protein): TNF-a, 15.2 to 58.414; IL-1b, 74.2 to 197.171; IL-6, 1.086 to 6.686; and MPO, 2.596 to 21.743. Post hoc analysis revealed that LT did not affect these factors in Sham groups but dose-dependently inhibited their increase in SCI groups (P < 0.001), with 10 mg/kg and

Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009

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Figure 2 LT treatment prevents post-SCI reduction of spinal cord antioxidative strength. LT effects on the protective enzymes SOD and CAT in rat spinal cord after SCI were measured. Data represent the mean  SEM (N Z 7). Treatment groups: SCI: SCI þ saline; SCI þ T10: SCI þ LT 10 mg/kg; SCI þ T30: SCI þ LT 30 mg/kg; Sham: laminectomy þ saline; Sham þ T10: laminectomy þ LT 10 mg/kg; Sham þ T30: laminectomy þ LT 30 mg/kg; C: control. ***P < 0.001 vs. C, ###P < 0.001 vs. SCI; data were analyzed by a mixed repeated ANOVA and post hoc using one-way ANOVA with Tukey pairwise tests.

30 mg/kg LT producing the following reductions (respectively) in levels of the factors (in nmol/mg protein): TNF-a, 58.414 to 48.543 (16.9%) and 58.414 to 36.657 (37.25%); IL1b, 197.171 to 162.886 (17.39%) and 197.171 to 125.543 (36.33%); IL-6, 6.686 to 4.899 (26.73%) and 6.686 to 3.897 (41.71%); and MPO, 21.743 to 17.599 (19.06%) and 21.743 to 11.971 (44.94%). These results suggest that LT suppressed the neuroinflammation and neutrophil infiltration that were increased post-SCI in rat spinal cord.

P < 0.001)] at 72-h post-SCI. Post hoc analysis results showed that LT exerted no effect on caspase-3 in Sham groups but dose-dependently suppressed caspase-3 elevations in SCI groups (P < 0.001), with 10 mg/kg and 30 mg/kg LT reducing caspase-3 (respectively) by 18.17% and 34.94% (from 55.686 to 45.571 nmol/mg and 55.686 to 36.229 nmol mg protein). These results suggest that LT inhibited the apoptosis enhanced after SCI in rat spinal cord.

LT inhibits post-SCI increases in apoptosis in the spinal cord

LT improves recovery of behavioral motor functions after SCI

We evaluated how LT affects apoptosis by measuring caspase-3 in the spinal cord after SCI (Fig. 4). Caspase-3 level in the SCI group was significantly increased [from 10.714 to 55.686 nmol/mg protein (t-test, C vs. SCI;

We then examined how LT affects behavioral motor functions after SCI (Fig. 5). Hindlimb behavioral motor functions were markedly impaired in rats post-SCI. Immediately after SCI, rats in all groups showed paralysis of both hindlimbs.

Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009

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neuroprotection in spinal cord injury

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Figure 3 LT treatment reduces increases in spinal cord neuroinflammation and neutrophil accumulation after SCI. LT effects on TNF-a, IL-1b, IL-6, and MPO levels in the spinal cord after SCI were measured; data represent the mean  SEM (N Z 7). Treatment groups: SCI: SCI þ saline; SCI þ T10: SCI þ LT 10 mg/kg; SCI þ T30: SCI þ LT 30 mg/kg; Sham: laminectomy þ saline; Sham þ T10: laminectomy þ LT 10 mg/kg; Sham þ T30: laminectomy þ LT 30 mg/kg; C: control. ***P < 0.001 vs. C, ###P < 0.001, ## P < 0.01 vs. SCI; data were analyzed by a mixed repeated ANOVA and post hoc using one-way ANOVA with Tukey pairwise tests.

Scores for BBB rating, toe-spread, and hindfoot bar-grab tests were 0, and the maximum degree measured in the inclined-plane test was w80 . Although motor functions gradually improved during the 14 days of the experiment,

the test scores were lower (P < 0.001) in the SCI group than in the Sham group (Fig. 5). Notably, 10 mg/kg and 30 mg/kg LT treatment led to significant elevations of all four test scores starting at 1-week post-SCI (Fig. 5), with behavioral

Figure 4 LT treatment lowers post-SCI increases in apoptosis in spinal cord. LT effect on caspase-3 level in rat spinal cord postSCI was measured. Data represent the mean  SEM (N Z 7). Treatment groups: SCI: SCI þ saline; SCI þ T10: SCI þ LT 10 mg/kg; SCI þ T30: SCI þ LT 30 mg/kg; Sham: laminectomy þ saline; Sham þ T10: laminectomy þ LT 10 mg/kg; Sham þ T30: laminectomy þ LT 30 mg/kg; C: control. ***P < 0.001 vs. C, ###P < 0.001, ##P < 0.01 vs. SCI; data were analyzed by a mixed repeated ANOVA and post hoc using one-way ANOVA with Tukey pairwise tests.

Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009

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Figure 5 LT treatment enhances recovery of behavioral motor functions after SCI. We measured LT effects on post-SCI recovery in (A) BBB locomotor-rating scale, (B) inclined-plane, (C) toe-spread, and (D) hindfoot bar-grab tests. Data represent the mean  SEM (N Z 7). Treatment groups: SCI: SCI þ saline; SCI þ T10: SCI þ LT 10 mg/kg; SCI þ T30: SCI þ LT 30 mg/kg; Sham: laminectomy þ saline; Sham þ T10: laminectomy þ LT 10 mg/kg; Sham þ T30: laminectomy þ LT 30 mg/kg; C: control. ***P < 0.001 vs. C, ###P < 0.001 vs. SCI; data were analyzed by a mixed repeated ANOVA and post hoc using one-way ANOVA with Tukey pairwise tests.

motor-function recovery accelerated starting from this point after LT treatment. At 14-days post-injury, most rats in the SCI group showed minimal movement of the two joints and no plantar stepping, partial toe spreading, or hindfoot response to bar contact, as well as a lack of ability to grab the bar. By contrast, rats treated with 10 mg/kg and 30 mg/kg LT showed occasional weight-supported plantar steps and frequent forelimb-hindlimb coordination, improved toe spreading to more than the partial spreading state, and the ability to grab the bar with the hindfoot. Moreover, the maximum degree in the inclined-plane test reached 60 . Relative to the SCI group, LT (30) groups showed significant increases in the scores of the BBB rating test (11.143  1.345 vs. 19.857  0.488; P < 0.001) (Fig. 5A), inclined-plane test (62.143  2.41 vs. 70.857  3.625 ; P < 0.001) (Fig. 5D), toe-spread test (2.4  0.258 vs. 2.943  0.053; P < 0.05) (Fig. 5B), and hindfoot bar-grab test (2.129  0.18 vs. 2.871  0.111; P < 0.05) (Fig. 5C).

Discussion Our results showed that LT exerted a protective effect against SCI in rats. LT treatment reduced the extent of post-traumatic oxidative/nitrosative stresses, inflammation, and apoptosis in the spinal cord after SCI, and LT

treatment also promoted post-SCI recovery of behavioral motor functions in rats. These results suggest that LT can produce therapeutic effects, potentially through its potent antioxidative, anti-inflammatory, and antiapoptotic activities, and thereby improve functional outcomes after SCI. Several experimental models have been developed to induce the major events of SCI in rats; however, compression injury, a rapid insult produced by an aneurysm clip to the dura through a four-level (T5eT8) laminectomy, which results in edema and tissue injury associated with motorfunction loss, is the most widely used animal SCI model.3,25,26 As in previous studies, we found that rats showed paralysis of both hindlimbs immediately after SCI and exhibited high oxidative-/nitrosative-stress states, neuroinflammation, and apoptosis in spinal cord tissue. Notably, our results demonstrated that LT enhanced postSCI locomotor recovery. We are the first group to report that LT treatment drastically promotes the recovery of behavioral motor functions after SCI in this animal model. These findings indicate that LT enhances the extent of recovery from spinal cord damage. After spinal cord tissue is injured, neutrophils and other phagocytes reach the injury site and produce hypochlorite, a strong oxidant synthesized by MPO, an enzyme present in neutrophil granules. MPO activity strongly corresponds with the number of infiltrated neutrophils in lesion sites and their inflammatory activity in injured areas.27 Typically,

Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009

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neuroprotection in spinal cord injury

MPO activity is increased within 3 h and remains elevated for up to 3 days after SCI.3,7,28 MPO catalyzes the reaction between H2O2 and chloride ions that produces hypochlorite, which reacts with polyunsaturated fatty acids and causes lipid peroxidation. Moreover, neutrophils and macrophages play a major role in oxidative stress by generating reactive oxygen species, which causes cytotoxicity by oxidizing lipids, proteins, and nucleic acids in injured spinal cord tissue.29,30 Because oxidative stress has major implications for the development of spinal cord lesions after primary injury, and given that polyunsaturated fatty acids are abundant in the spinal cord, increased susceptibility to free radical damage would be expected to elevate oxidative stress. Lipid peroxidation is initiated as early as 5 min post-SCI and continues for the first week after injury. In agreement with previous studies, we found that MDA, the major product of polyunsaturated fatty acid oxidation and a reliable biomarker of oxidative injury, and MPO were increased, whereas SOD and CAT were decreased, at 72-h post-SCI, demonstrating susceptibility to enhanced oxidative stress, neutrophil accumulation, and inflammatory reaction in rats after SCI.3,7,25,26,31 Inflammatory response is a crucial process in tissuedamage development after SCI. Within a few hours after experimental SCI, inflammatory cytokines are upregulated and remain elevated for up to 3 days post-SCI.3,5,7,17 In the present study, we demonstrated that TNF-a, IL-1b, and IL-6 levels were elevated at 72-h post-SCI, suggesting the involvement of inflammatory responses after SCI. Elevated levels of inflammatory cytokines mediate inflammation, including induction of other cytokines, oxidant production, stimulation of astrocyte proliferation, glial-cell activation, promotion of neuronal apoptosis, and irreversible pathological changes, thereby aggravating SCI. Additionally, overexpression of inflammatory cytokines might be central to neurodegeneration due to their early production after SCI.3,5,7 NO is a diffusible, highly reactive gas produced by nitric oxide synthase (NOS), which is mostly expressed in astrocytes and microglial cells. NO and NOS are critically involved in central nervous system (CNS) injury. Physiologically, NO is produced in small amounts by endothelial and neuronal cells in the CNS, whereas NO levels increase considerably immediately after SCI. The high concentration of NO produced by inducible NOS (iNOS) reacts with O2 to form the reactive oxidant peroxynitrite (ONOO), which can directly oxidize lipids, proteins, and DNA and can itself cause neuronal loss and locomotor dysfunction in SCI. Moreover, excessive NO can induce apoptosis, a critical factor in SCI secondary changes.3,28,30,32 In the present study, we observed that NO levels were increased at 72-h post-SCI, suggesting that nitrosative stress can also influence the development of behavioral motor dysfunctions after SCI. Inflammatory reactions reportedly cause increased iNOS expression and lead to excessive NO production after SCI.33,34 Collectively, these findings support the notion that both oxidative/nitrosative stresses and inflammatory responses participate in the development of post-SCI impairment of behavioral motor functions. Activation of inflammatory responses and oxidative/ nitrosative stresses also damage cells and induce apoptotic cascades after SCI. Apoptosis causes the death of neurons

9 and oligodendrocytes in the spine and further agitates and damages the axon-myelin unit and diminishes impulse conduction, which result in neuronal loss.2,3,5 Long-term deficits in behavioral motor function after SCI are considered to result from widespread apoptosis of neurons and oligodendrocytes in regions distant from the primary injury site.4,28 In agreement with previous findings, our study showed that caspase-3 was upregulated at 72-h post-SCI, suggesting that apoptotic cascades were activated after SCI. Apoptosis occurs initially at 6 h after SCI at the lesion epicenter, and for several days thereafter, the number of apoptotic cells rises steadily.3,5,28 Our results suggested that in rats in which motor functions were impaired, oxidative-/nitrosative-stress states, neuroinflammation, and apoptosis were elevated in the spinal cord after SCI. This offers a favorable therapeutic target: lowering excessive free radicals and NO, increasing antioxidative factors, or blocking inflammatory and apoptotic cascades might help enhance recovery of motor functions after SCI. LT reportedly exerts strong antioxidative and anti-inflammatory effects10,11,14e16; therefore, LT might possess the properties necessary to reduce post-SCI oxidative damage, neuroinflammation, and apoptosis in rat spinal cord. We found that in the rat spinal cord, LT dose-dependently reduced SCI-enhanced levels of MDA, NO, MPO, TNF-a, IL-1b, IL-6, and caspase-3 and increased SCI-reduced SOD and CAT levels, suggesting that the multiple functions of LT are linked to several pathophysiological pathways, and that these properties might underpin the mechanisms by which LT improves post-SCI recovery of motor functions, as indicated by our behavioral analyses. Other studies show that the development of post-SCI motor-function impairment can be prevented by the protective effects of enzymes, such as SOD and CAT2,3,8,25,31 and by the suppression of inflammatory cytokines.2,4,5,7,8,28 Therefore, the results showing LTinduced motor-function improvement post-SCI can be attributed at least partly to the ability of LT to clear excessive free radicals and NO and inhibit inflammation and apoptotic cascades in the rat spinal cord after SCI. A limitation of our study is that LT clinical efficacy (including optimal therapeutic dose and treatment time) could not be determined, because an experimental SCI animal model was used. Moreover, further investigation is required to elucidate the mechanisms underlying LT-specific protective effects in SCI, and clinical studies must be conducted to confirm LT safety and validate our findings. Nevertheless, considering the results of this study, LT might represent a candidate for the development of therapeutic agents for SCI. In summary, LT administration was found to 1) inhibit increases in NO and MDA levels, 2) prevent decreases in antioxidant activity, 3) lower levels of neuroinflammatory and apoptotic markers in the rat spinal cord after SCI, and, notably, 4) promote post-SCI recovery of behavioral motor functions in rats. Although the mechanisms underlying LT protection against SCI in rats require further elucidation, our findings provide evidence of LT-specific therapeutic potential for SCI treatment in animal models. This efficacy potential probably derives from the strong antioxidative, anti-inflammatory, and antiapoptotic activities of LT, which could presumably also help improve functional outcomes during the treatment of clinically relevant human SCI.

Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009

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Funding This study was supported by Mackay Memorial Hospital (MMH-106-76,77). We would like to thank Editage (www. editage.com) for English-language editing.

Declaration of Competing Interest The authors declare that they have no conflict of interest.

References 1. Akbari M, Khaksari M, Rezaeezadeh-Roukerd M, Mirzaee M, Nazari-Robati M. Effect of chondroitinase ABC on inflammatory and oxidative response following spinal cord injury. Iran J Basic Med Sci 2017;20:806e12. 2. Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995;12:1e21. 3. Berker KI1, Ozdemir Olgun FA, Ozyurt D, Demirata B, Apak R. Modified Folin-Ciocalteu antioxidant capacity assay for measuring lipophilic antioxidants. J Agric Food Chem 2013;61: 4783e91. 4. Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Investig Dermatol 1982;78:206e9. 5. Cemil B1, Topuz K, Demircan MN, Kurt G, Tun K, Kutlay M, et al. Curcumin improves early functional results after experimental spinal cord injury. Acta Neurochir 2010;152:1583e90. 6. Chen CN, Chang KC, Wang MH, Tseng HC, Soung HS. Protective effect of L-theanine on haloperidol-induced orofacial. Chin J Physiol 2018;61:35e41. 7. Cong L, Chen W. Neuroprotective effect of ginsenoside Rd in spinal cord injury rats. Basic Clin Pharmacol Toxicol 2016;119: 193e201. 8. Do gruer ZN1, Unal M, Eskandari G, Pata YS, Akbas‚ Y, Cevik T, et al. Malondialdehyde and antioxidant enzymes in children with obstructive adenotonsillar hypertrophy. Clin Biochem 2004;37:718e21. 9. Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: Malonaldehyde and 4-hydroxynonenal. Methods Enzymol 1990;186:407e21. 10. Farahabadi A, Akbari M, Amini Pishva A, Zendedel A, Arabkheradmand A, Beyer C, et al. Effect of progesterone therapy on TNF-a and iNOS gene expression in spinal cord injury model. Acta Med Iran 2016;54:345e51. 11. Hall ED, Wang JA, Bosken JM, Singh IN. Lipid peroxidation in brain or spinal cord mitochondria after injury. J Bioenerg Biomembr 2016;48:169e74. 12. Hou QX, Yu L, Tian SQ, Jiang CJ, Yang WJ, Wang ZJ. Neuroprotective effects of atomoxetine against traumatic spinal cord injury in rats. Iran J Basic Med Sci 2016;19:272e80. 13. Jamwal S, Kumar P. L-theanine, a component of green tea prevents 3-nitropropionic acid (3-NP)-induced striatal toxicity by modulating nitric oxide pathway. Mol Neurobiol 2017;54: 2327e37. 14. Jamwal S, Singh S, Gill JS, Kumar P. L-theanine prevent quinolinic acid induced motor deficit and striatal neurotoxicity: reduction in oxido-nitrosative stress and restoration of striatal neurotransmitters level. Eur J Pharmacol 2017;811:171e9. 15. Karsy M, Hawryluk G. Pharmacologic management of acute spinal cord injury. Neurosurg Clin N Am 2017;28:49e62.

C.-C. Yang et al. 16. Kerasidis H, Wrathall JR, Gale K. Behavioral assessment of functional deficit in rats with contusive spinal cord injury. J Neurosci Methods 1987;20:167e79. 17. Kermani HR, Nakhaee N, Fatahian R, Najar AG. Effect of aspirin on spinal cord injury: an experimental study. Iran J Med Sci 2016;41:217e22. 18. Kim DK, Kweon KJ, Kim P, Kim HJ, Kim SS, Sohn NW, et al. Ginsenoside Rg3 improves recovery from spinal cord injury in rats via suppression of neuronal apoptosis, pro-inflammatory mediators, and microglial activation. Molecules 2017;22:E122. 19. Kopper TJ, Gensel JC. Myelin as an inflammatory mediator: myelin interactions with complement, macrophages, and microglia in spinal cord injury. J Neurosci Res 2018;96:969e77. 20. Kurt G, Ergu ¨rcek AO, Bo ¨rcek P, Gu ¨n E, Cemil B, Bo ¨lbahar O, et al. Neuroprotective effects of infliximab in experimental spinal cord injury. Surg Neurol 2009;71:332e6. 21. Liu C, Wu W, Zhang B, Xiang J, Zou J. Temporospatial expression and cellular localization of glutamine synthetase following traumatic spinal cord injury in adult rats. Mol Med Rep 2013;7:1431e6. 22. Amini Pishva A, Akbari M, Farahabadi A, Arabkheradmand A, Beyer C, Dashti N, et al. Effect of estrogen therapy on TNF-a and iNOS gene expression in spinal cord injury model. Acta Med Iran 2016;54:296e301. 23. Rouanet C, Reges D, Rocha E, Gagliardi V, Silva GS. Traumatic spinal cord injury: current concepts and treatment update. Arq Neuropsiquiatr 2017;75:387e93. 24. Soung HS, Wang MH, Chang KC, Chen CN, Chang Y, Yang CC. LTheanine decreases orofacial dyskinesia induced by reserpine in rats. Neurotox Res 2018;34:375e87. 25. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497e500. 26. Taskiran D, Kutay FZ, Sozmen E, Pogun S. Sex differences in nitrite/nitrate levels and antioxidant defense in rat brain. Neuroreport 1997;8:881e4. 27. Tu ¨zu ¨rko ¨ D, S‚anlier N. L-theanine, unique amino acid of tea, and its metabolism, health effects, and safety. Crit Rev Food Sci Nutr 2017;57:1681e7. 28. Visavadiya NP, Patel SP, VanRooyen JL, Sullivan PG, Rabchevsky AG. Cellular and subcellular oxidative stress parameters following severe spinal cord injury. Redox Biol 2016; 8:59e67. 29. Wang D, Gao Q, Wang T, Qian F, Wang Y. Theanine: the unique amino acid in the tea plant as an oral hepatoprotective agent. Asia Pac J Clin Nutr 2017;26:384e91. 30. Xun C, Mamat M, Guo H, Mamati P, Sheng J, Zhang J, et al. Tocotrienol alleviates inflammation and oxidative stress in a rat model of spinal cord injury via suppression of transforming growth factor-b. Exper Ther Med 2017;14:431e8. 31. Yin C, Guo L, Liu Y, Yin X, Zhang L, Jia G, et al. Antidepressantlike effects of L-theanine in the forced swim and tail suspension tests in mice. Phytother Res 2011;25:1636e9. 32. Yin X, Yin Y, Cao FL, Chen YF, Peng Y, Hou WG, et al. Tanshinone IIA attenuates the inflammatory response and apoptosis after traumatic injury of the spinal cord in adult rats. PLoS One 2012;7:e38381. 33. Yoto A, Motoki M, Murao S, Yokogoshi H. Effects of L-theanine or caffeine intake on changes in blood pressure under physical and psychological stresses. J Physiol Anthropol 2012;31:28. 34. Zhang D, Ma G, Huo M, Zhang T, Chen L, Zhao C. The neuroprotective effect of puerarin in acute spinal cord injury rats. Cell Physiol Biochem 2016;39:1152e64. 35. Zheng G, Sayama K, Okubo T, Juneja LR, Oguni I. Anti-obesity effects of three major components of green tea, catechins, caffeine and theanine, in mice. In Vivo 2004;18:55e62.

Please cite this article as: Yang C-C et al., L-Theanine improves functional recovery after traumatic spinal cord injury in rats, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.11.009