科技部補助專題研究計畫報告
延續前年計畫: 利用年老小鼠中風模式探討褪黑激素結合
agomelatine於中風後神經保護與神經重塑機制上扮演的角色
(II-III)(第2年)
報 告 類 別 ： 成果報告 計 畫 類 別 ： 個別型計畫 計 畫 編 號 ： MOST 107-2314-B-006-027-MY2 執 行 期 間 ： 108年08月01日至109年07月31日 執 行 單 位 ： 國立成功大學醫學系外科 計 畫 主 持 人 ： 李宜堅 計畫參與人員： 學士級-專任助理：林士傑本研究具有政策應用參考價值：■否 □是，建議提供機關
（勾選「是」者，請列舉建議可提供施政參考之業務主管機關）
本研究具影響公共利益之重大發現：□否 □是
中 華 民 國 109 年 10 月 28 日
中 文 摘 要 ： 流行病學的統計指出臨床上腦中風好發於年老的個體上，且在中風 後的死亡率高且恢復力差。顯示出老化對於腦中風而言是重要的危 險因子。目前針對腦中風研究的動物模型，多採用年輕動物來進行 研究，不符合臨床上的情況。因此，以老動物做動物實驗更有其臨 床意義。褪黑激素是一種強效的抗氧化劑與自由基截取劑。我們的 研究顯示它具有減少腦缺血後腦梗塞體積能力及增強受傷腦細胞之 存活以及讓腦神經電生理傳導並感覺運動功能回復的作用。本計畫 旨在探討褪黑激素在年老個體上對於缺血性腦中風下之腦神經保護 與神經重塑的效能為何？ 過去我們的研究顯示，在中風模型的老鼠上施打褪黑激素(5 mg/kg)所造成腦神經的電性傳導與感覺運動功能的恢復，其可能路 經之一是經由NMDA receptor與PSD-95蛋白造成神經重塑功能增強所 致。已知褪黑激素會刺激MT1與MT2 receptor。但有研究顯示褪黑激 素所造成的神經保護功能不經由MT1與MT2 receptor。然而褪黑激素 造成的神經重塑功能增強是否經由MT1與MT2 receptor仍不清楚。在 本計畫中我們設計一系列活體之年輕與年老個體永久性缺血中風模 式以及離體培養神經細胞，來探討褪黑激素之神經再塑能力在年輕 與年老的老鼠的差異，並分析所媒介路徑接受器與下游路徑為何。 在動物實驗中，ICR小鼠於中大腦動脈進行永久性栓塞後給予褪黑激 素(5mg/kg)，並且在手術一天後，進行神經行為評估，觀察其感覺 與運動功能恢復情況，同時犧牲老鼠取出大腦以Golgi-Cox stain及 其他組織染色評估中風誘發之神經元樹突傷害及重塑的差異性。 實驗結果證實褪黑激素在老化個體上，在缺血性腦中風情況下仍然 能夠降低缺血傷害並增加感覺運動功能恢復。此外藉由Golgi-cox stain的觀察結果得知，在栓塞同側之缺血半影區內的腦皮質II-III層與腦皮質III-IV層以及同側腦皮質V-VI層褪黑激素處理組比控 制組樹突小刺密度有較顯著的增加。而在同側缺血半影區內的腦皮 質II-III層與腦皮質III-IV層褪黑激素處理組比控制組樹突分支有 較顯著的複雜性。另外，在對側的腦皮質V-VI層褪黑激素褪黑激素 處理組比控制組樹突小刺密度有較顯著的增加。在免疫染色與西方 墨點法分析，發現褪黑激素在年老個體上於缺血中風情況之下，仍 然能夠降低缺血傷害和增加行為學的恢復。我們的結果亦指出褪黑 激素可透過BDNF來調控GAP-43 (growth- associated protein 43)與PSD-95 (post-synaptic density-95)表現，這些參與神經細 胞的分化、成長，是神經生長發育和受損修復時不可缺少的重要蛋 白。 於離體細胞實驗神經細胞褪黑激素 20 M 和50 M，我們同時利用褪 黑激素作用在培養的神經細胞上，探討其誘發神經塑性的分子路徑 。實驗結果顯示褪黑激素會造成腦細胞的分支與樹突小刺 中 文 關 鍵 詞 ： 年老的個體、腦中風、褪黑激素 、神經再塑。
英 文 摘 要 ： Epidemiological studies have shown that age is an important determinant regarding the occurrence rate of stroke.
Moreover, the mortality rate and prognosis were even worse in subjects of elder. Nevertheless, most studies on animal models of stroke were performed on young animals and, therefore, the results are less clinically applicable to
human subjects. Using aged animals in the studies of stroke is more clinical application than the young ones. Melatonin is a well-known potent free radical scavenger and an
antioxidant. The purpose of this study is to assess the impact of age effect on the benefit of neuroplasticity after the treatment of melatonin in ischemia models in vivo and in vitro.
Our studies have also revealed that the benefit in electrical signal transmission in neurons and
neurobehavioral performance after intravenous injection with melatonin (5 mg/kg) might have mediated through NMDA receptor and PSD-95. Although MT1 and MT2 were the target receptors of melatonin, recent data have shown that the melatonin induced neuroprotective effect was MT1 and MT2 receptors independent. Whether the melatonin-induced neuroplasticity is MT1 and MT2 receptors dependent was still a question to be answered. In this project, we conduct a series of surveys to discriminate melatonin's effect on young and elder mice after brain insult. The role of MT1 and MT2 is also studied. ICR mice were subjected to permanent brain ischemia by MCA ligation surgery, followed by melatonin treatment (5mg/kg, iv. injection). The
surveillance of neurobehavioral was conducted the next day. The brain was harvested for quantitative studies of
melatonin effect on dendritic spines and the density after brain insult by Golgi-Cox stain and histochemistry.
Our data revealed that both young and aged mice exhibited a significant reduction in the brain's infarction volumes and improved neuronal behavioral outcomes as accessed by
sensorimotor and rotarod motor performance tests when treated with melatonin. In the results of Golgi-cox stain, melatonin treatment resulted in significantly higher
dendritic spine density in the second-, third-order basilar dendrites of the pyramidal cells in the layer II-III and layer III-IV of the penumbra, the layer V-VI of the ischemic cortex, and the layer V-VI in contralateral cortex, as compared to the vehicle-injected controls. Moreover, melatonin treatment led to better dendritic
arborization in the pyramidal cells in the layer II-III and layer III-IV of the penumbra of the ischemic cortex
compared to the vehicle-injected control. Melatonin also significantly improved the BDNF, GAP-43, and SNAP-25 protein expression in the ischemic brain, influential in the regeneration and repairment during brain damage.
Our in vitro data have shown that pretreatment of melatonin (20 and 50 µM) significantly increased the dendritic
density and development in cultured neurons and concomitant increases of BDNF, GAP-43, and PSD-95 protein expression.
Elevation and inhibition of M1, M2 receptors of cultured neurons by the agonist (Ramelteon) and antagonist
(Luzindole) have shown an induction and suppression of melatonin induced BDNF expression. As BDNF is the principle regulator of dendritic development, melatonin might induce neuroplasticity through M1, M2 receptors.
英 文 關 鍵 詞 ： Melatonin, aged-permanent ischemia models, and neuroplasticity
I
科技部補助專題研究計畫成果報告
（□期中進度報告/¢期末報告）
（計畫名稱
） 中文:延續前年計畫: 利用年老小鼠中風模式探討褪黑激素結合 agomelatine 於中風後神經保護與神經 重塑機制上扮演的角色 (II-III)英文:Neuroplastic and neuroprotective effects of the combined use with melatonin and agomelatine in aged mice subjected to focal cerebral ischemia (II-III)
計畫類別：□個別型計畫 □整合型計畫
計畫編號：MOST 107-2314-B-006-027-MY2
執行期間：107 年 08 月 01 日 至 109 年 07 月 31 日
執行機構及系所：國立成功大學醫學系外科
計畫主持人
：李宜堅 教授
共同主持人
：
計畫參與人員
：林士傑 學士級專任助理
本計畫除繳交成果報告外
，另含下列出國報告，共 份：
□
執行國際合作與移地研究心得報告
□
出席國際學術會議心得報告
□
出國參訪及考察心得報告
1
目錄
(一) 計畫中文摘要 ... 2
(二) 計畫英文摘要 ... 3
前言及文獻探討
... 4
研究目的
... 5
Material and Methods ... 6
Results ... 9
Conclusion ... 28
References ... 29
科技部補助專題研究計畫成果報告自評表
... 31
2 (一) 計畫中文摘要 流行病學的統計指出臨床上腦中風好發於年老的個體上，且在中風後的死亡率高且恢復力差。顯示 出老化對於腦中風而言是重要的危險因子。目前針對腦中風研究的動物模型，多採用年輕動物來進行 研究，不符合臨床上的情況。因此，以老動物做動物實驗更有其臨床意義。褪黑激素是一種強效的抗 氧化劑與自由基截取劑。我們的研究顯示它具有減少腦缺血後腦梗塞體積能力及增強受傷腦細胞之存 活以及讓腦神經電生理傳導並感覺運動功能回復的作用。本計畫旨在探討褪黑激素在年老個體上對於 缺血性腦中風下之腦神經保護與神經重塑的效能為何？ 過去我們的研究顯示，在中風模型的老鼠上施打褪黑激素(5 mg/kg)所造成腦神經的電性傳導與感覺 運動功能的恢復，其可能路經之一是經由NMDA receptor 與 PSD-95 蛋白造成神經重塑功能增強所致。 已知褪黑激素會刺激MT1 與 MT2 receptor。但有研究顯示褪黑激素所造成的神經保護功能不經由 MT1 與MT2 receptor。然而褪黑激素造成的神經重塑功能增強是否經由 MT1 與 MT2 receptor 仍不清楚。在 本計畫中我們設計一系列活體之年輕與年老個體永久性缺血中風模式以及離體培養神經細胞，來探討 褪黑激素之神經再塑能力在年輕與年老的老鼠的差異，並分析所媒介路徑接受器與下游路徑為何。在 動物實驗中，ICR 小鼠於中大腦動脈進行永久性栓塞後給予褪黑激素(5mg/kg)，並且在手術一天後，進 行神經行為評估，觀察其感覺與運動功能恢復情況，同時犧牲老鼠取出大腦以Golgi-Cox stain 及其他 組織染色評估中風誘發之神經元樹突傷害及重塑的差異性。 實驗結果證實褪黑激素在老化個體上，在缺血性腦中風情況下仍然能夠降低缺血傷害並增加感覺運
動功能恢復。此外藉由Golgi-cox stain 的觀察結果得知，在栓塞同側之缺血半影區內的腦皮質 II-III 層
與腦皮質III-IV 層以及同側腦皮質 V-VI 層褪黑激素處理組比控制組樹突小刺密度有較顯著的增加。而
在同側缺血半影區內的腦皮質II-III 層與腦皮質 III-IV 層褪黑激素處理組比控制組樹突分支有較顯著的
複雜性。另外，在對側的腦皮質V-VI 層褪黑激素褪黑激素處理組比控制組樹突小刺密度有較顯著的增
加。在免疫染色與西方墨點法分析，發現褪黑激素在年老個體上於缺血中風情況之下，仍然能夠降低
缺血傷害和增加行為學的恢復。我們的結果亦指出褪黑激素可透過BDNF 來調控 GAP-43 (growth-
associated protein 43)與 PSD-95 (post-synaptic density-95)表現，這些參與神經細胞的分化、成長，是神 經生長發育和受損修復時不可缺少的重要蛋白。 於離體細胞實驗神經細胞褪黑激素 20 µM 和 50 µM，我們同時利用褪黑激素作用在培養的神經細 胞上，探討其誘發神經塑性的分子路徑。實驗結果顯示褪黑激素會造成腦細胞的分支與樹突小刺密度 增加，我們同時看到在培養的神經細胞上，褪黑激素造成BDNF、SNAP-25 與 PSD-95 的表現量增加。 顯示褪黑激素是透過BDNF 引起下游的訊息反應，在促成型態的改變。當我們以褪黑激素的刺激劑 Ramelteon 與拮抗劑 Luzindole 去處理褪黑激素刺激過的神經細胞，發現他分別可以造成 BDNF 的上升 與下降。由於BDNF 是造成神經塑性的主要路徑，因此我們的研究顯示，褪黑激素可能是經過 MT1, MT2 受體造成BDNF 上升，及部分或全部 BDNF 的路徑，來修復神經可塑性的功能。 中文關鍵詞： 年老的個體、腦中風、褪黑激素和神經再塑。
3
(二) 計畫英文摘要
Epidemiological studies have shown that age is an important determinant regarding the occurrence rate of stroke. Moreover, the mortality rate and prognosis were even worse in subjects of elder. Nevertheless, most studies on animal models of stroke were performed on young animals and, therefore, the results are less clinically applicable to human subjects. Using aged animals in the studies of stroke is more clinical
application than the young ones. Melatonin is a well-known potent free radical scavenger and an antioxidant. Our previous studies have demonstrated that melatonin treatment to young animals subjected to transient cerebral ischemia can reduce the infarction volume and lead to neuroplasticity, resulting in boosting the electrical signal transmission in neurons and enhancing neurobehavioral. The purpose of this study is to assess the impact of age effect on the benefit of neuroplasticity after the treatment of melatonin in ischemia models in vivo and in vitro.
Our studies have also revealed that the benefit in electrical signal transmission in neurons and neurobehavioral performance after intravenous injection with melatonin (5 mg/kg) might have mediated through NMDA receptor and PSD-95. Although MT1 and MT2 were the target receptors of melatonin, recent data have shown that the melatonin induced neuroprotective effect was MT1 and MT2 receptors independent. Whether the melatonin-induced neuroplasticity is MT1 and MT2 receptors dependent was still a question to be answered. In this project, we conduct a series of surveys to discriminate melatonin's effect on young and elder mice after brain insult. The role of MT1 and MT2 is also studied. ICR mice were subjected to permanent brain ischemia by MCA ligation surgery, followed by melatonin treatment (5mg/kg, iv. injection). The
surveillance of neurobehavioral was conducted the next day. The brain was harvested for quantitative studies of melatonin effect on dendritic spines and the density after brain insult by Golgi-Cox stain and
histochemistry.
Our data revealed that both young and aged mice exhibited a significant reduction in the brain's infarction volumes and improved neuronal behavioral outcomes as accessed by sensorimotor and rotarod motor
performance tests when treated with melatonin. In the results of Golgi-cox stain, melatonin treatment resulted in significantly higher dendritic spine density in the second-, third-order basilar dendrites of the pyramidal cells in the layer II-III and layer III-IV of the penumbra, the layer V-VI of the ischemic cortex, and the layer V-VI in contralateral cortex, as compared to the vehicle-injected controls. Moreover, melatonin treatment led to better dendritic arborization in the pyramidal cells in the layer II-III and layer III-IV of the penumbra of the ischemic cortex compared to the vehicle-injected control. Melatonin also significantly improved the BDNF, GAP-43, and SNAP-25 protein expression in the ischemic brain, influential in the regeneration and
repairment during brain damage.
Our in vitro data have shown that pretreatment of melatonin (20 and 50 µM) significantly increased the dendritic density and development in cultured neurons and concomitant increases of BDNF, GAP-43, and PSD-95 protein expression. Elevation and inhibition of M1, M2 receptors of cultured neurons by the agonist (Ramelteon) and antagonist (Luzindole) have shown an induction and suppression of melatonin induced BDNF expression. As BDNF is the principle regulator of dendritic development, melatonin might induce neuroplasticity through M1, M2 receptors.
4
計畫內容
前言及文獻探討
Age is one important risk factor for cerebral ischemia because the recovery after stroke is significantly influenced by age. One 75% of stroke victims are above 65-year-old, but their recovery is less than the middle-aged patients with stroke (1, 2). Post-stroke depression is of utmost clinical relevance in stroke survivors, either at the early or the late stage after stroke (3). Therefore, stroke in aged patients was most likely to suffer from higher morbidity, mortality, and grave functional outcome (4-6). Although numerous neuroprotective, anti-depression, anti-oxidative, or anti-inflammatory drugs show efficacy in the experimental animal models of stroke, none of these drugs are proven to be effective in clinical use. One of the key reasons for the failure of clinical trials is that experiment stroke models are often performed in healthy young adults rather than aged animals. In contrast, stroke predominantly occurs in the aged population (7). Therefore, using aged animals for the ischemic animal model is one critical issue for effectively translating experimental results for clinical use.
Recent studies have shown that neuronal differentiation is accompanied by increased levels of
synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptophysin (a calcium-binding protein found on presynaptic vesicle membranes) proteins in neurons (8). Besides, dendritic spine density has been implicated to play an essential role in neuroplasticity. The 43 kDa growth-associated protein (GAP-43) is a nervous
tissue-specific protein synthesized at high levels during axonal growth in neuronal development and axonal regrowth in regeneration in the peripheral and central nervous systems. GAP-43, therefore, represents an critical marker for axonal regeneration and sprouting. Accordingly, dendritic spine density and two presynaptic proteins, SNAP-25 and synaptophysin, and postsynaptic proteins PSD-95 were used as indices and outcome measures for neuronal plasticity in the study (9). Furthermore, it is well known that brain-derived neurotrophic factor (BDNF) is a prominent neurotrophic factor that contributes to neuronal survival, axonal branching, dendritic arborization, and synaptogenesis (9, 10). Mature BDNF activates Trk B signaling in which several downstream effectors, including Akt and ERK. Phosphorylated EKR and Akt regulate certain transcription factors and trigger gene expression and protein syntheses, such as GAP-43, PSD-95, and BDNF (10-12).
Melatonin (N-Acetyl-5-methoxytryptamine) has various pharmacological actions that may help treat ischemic stroke (13, 14). We have already shown that melatonin is a candidate of agent suited as one treatment modality in various ischemic stroke models with initial success. We have previously demonstrated that
exogenous administration with melatonin protects the brain against acute ischemic damage by reducing
oxidative damage and improving blood-brain barrier (BBB) permeability in protecting gray and white matter of the brain and functional neurovascular unit pathology (13-17). More recently, we have shown that melatonin improved the dendritic spine density and the somatosensory field potentials of the ischemic brain at the subacute stage of insult, and this was associated with an elevated level of SNAP-25, but not synaptophysin (a marker of synaptogenesis), protein expression (18). Although numerous researchers have shown that melatonin has pharmacological benefits for treating acute ischemic stroke, it remains to be determined for its possible role in developing neuroplasticity in the aged stroke model. For this purpose, we had established the aged-stroke model in vitro and in vivo. In this study, we examined whether melatonin reduced brain infarction and dendritic spine damage and improved neuroplasticity.
5
It has been shown that melatonin's neuroprotective effects against acute brain damage do not depend on melatonin receptors (M1 and M2) in a mouse model of ischemic stroke. Our preliminary data have shown that melatonin exhibited neuroprotection and neuroplasticity at the acute stage during brain ischemia. There is still a need to clarify if melatonin initiates neuroplasticity's signaling cascades through the receptor-dependent pathway. For this purpose, we have successfully developed an aged model of permanent focal cerebral ischemia in our lab. We will investigate the mechanism behind melatonin modulate neuroplasticity from the receptor side of view after stroke.
Specific aims
In the present study, we investigated melatonin's neuroplasticity mechanism in permanent ischemia stroke models of young- and aged- mice. We also investigated whether the neuroplasticity effect of melatonin is the melatonin receptor (MT1/MT2) dependent. Besides, we investigated whether melatonin treatment initiates neuroplasticity action and the signaling pathway at normal neuronal cells.
6
Material and Methods
Primary cortical neuronal culture
Cultured cortical cells were obtained from the cerebral cortices of 1-day-old Sprague-Dawley rats under pentobarbital anesthesia. The cortices were placed in ice-cold Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) and minced. Tissue chunks will then incubated in a papain solution (0.6 mg/ml papain and DNase I in HBSS) at 37℃for 30 min to dissociate the cells, and the reaction were terminated by adding
heat-inactivated horse serum. After the cell suspensions will centrifuged at 800 xg, pellets were plated onto poly-D-lysine-coated petri dishes. Dissociated cells were suspended in DMEM with 10% horse serum and incubated at 37℃in a humidified incubator with 5% CO2. Three hours after plating, the culture medium were replaced by a serum-free neurobasal medium containing 25 mM glutamate, 0.5 mM L-glutamine, and 2% B27 supplement (17504-044; Invitrogen Corp., Carisbad, CA, USA). Cultured cells were allowed to grow for approximately 6-8 days as young-cultured neurons and 13-15 days as aged-cultured neurons.
Animal preparation, anesthesia, and monitoring
The young (8–12 weeks, weighing 35-40 g) and aged (12-13 months, weighing 55-60 g) ICR male mice were supplied by the University Laboratory Animal Center, and were allowed free access to food and water. Animals were anesthetized with 1-2% halothane in 70% N2O/30% O2. During surgery, body temperature was maintained at 37.0±0.5°C, which corresponds to brain temperature of 37.2±0.5°C, using a thermostatically controlled heating blanket and rectal probe (Harvard Apparatus, South Natick, MA). The right femoral artery was cannulated for measuring arterial blood gases, glucose, hematocrit and blood pressure.
Experimental model of permanent focal cerebral ischemic
Distal permanent occlusion of right MCA was produced by electrocoagulation of the right distal MCA modified. This method enables us to make an occlusion of distal MCA. Briefly, a curved vertical incision approximately 3 mm in length were made between the animal's right orbit and external auditory canal. The temporalis muscle was deflected to allow access for the craniotomy, which were made 3 mm anterior and 1 mm lateral to the foramen ovale with a dental drill. The dura was incised and the right MCA exposed and coagulated approximately 2 mm proximal to the olfactory tract by electrocoagulation with fine bipolar forceps (Vetroson, Summit Hill Laboratories, Navesink, NJ). After coagulation, the artery was cut to avoid recanulization. During MCA occlusion, the ipsilateral LCBF at the ischemia core cortex were decreased to about 20% of baseline caused by MCA occlusion as determined by Laser-Doppler flowmetry. Body temperature were monitored continuously and maintained at 37.0±1.0℃during MCA occlusion.
Animal sacrifice and histologic outcome measure·
Animals were sacrificed under anesthesia and transcardiacly perfused with ice-cold phosphate-buffered saline (PBS; Molecular Probes, Eugene, OR) and 4% paraformaldehyde. The brain was quickly removed, dehydrated in 15% and 30% sucrose sequentially, and then embedded in Optimal Cutting Temperature compound (OCT, Miles Inc., Elkhart, IN) and frozen in liquid nitrogen. Animals’ brains were sectioned coronally on a cryostat (HM-500O, Microm International GmbH, Walldorf, Germany). Serial sections of 40 μm at the preselected coronal levels, with 1-mm interval from the stereotaxic coordinates of the Bregma AP +4.22
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to –6.78 mm, were mounted on poly-L-lysine coated slides and dried at 37°C overnight. Sections (40 μm) were stained with 0.5% cresyl violet. Areas of brain infarction were measured using a computerized image analyzer (MCID Elite, Imaging Research Inc., St. Catherines, Ontario, Canada). Infarct volumes were expressed as a percentage of the contralateral hemisphere volume. In addition, individual cortical and subcortical
(caudoputaminal and hippocampal) infarct sizes and surviving neurons will separately be calculated.
Quantification of gray matter damage
One set of sections were stained with 0.5% cresyl violet. Under light microscopy, the areas of neuronal perikarya displaying typical morphological features of ischemic damage were delineated. Brain
infarction were measured using a computerized image analyzer (MCID Elite, Imaging Research Inc., Ontario, Canada), and were expressed as a percentage of the contralateral hemisphere volume. In addition, individual cortical and subcortical (caudoputaminal and hippocampal) infarct sizes were separately calculated.
Golgi–Cox analysis
At Day 3 and 7, animals were sacrificed, and the brains are rapidly removed and immersed in a modified Golgi–Cox solution for 14 days and, then, in 30% sucrose solution for another 5 days. Coronal brain sections of 200 μm were harvested using a vibratome (VSLM1 motorized Vibroslice, Campden Instrument, Silbey, UK), mounte, and stain. The spine density of the second- and third-order basilar dendrites of the pyramidal neurons at regions corresponding to the ischemic core (layer II-III), the inner (layer III-IV) and the outer (layer V-VI) boundary zones of the forepaw and the hindpaw areas of the ischemic hemisphere, as well as at the layer V-VI region of the contralateral, intact brain, were quantified and traced at 1000 magnification by a Zeiss Axioskop 2 Mot fluorescent microscope equipped with an Axiocam high-resolution camera and AxioVsion image analysis system (Carl Zeiss, Oberkochen, Germany).
Western blotting of BDNF, GAP-43, PSD-95, and SNAP-25
In in vivo model, samples were obtained from brain tissues of the ischemic core and penumbral regions which are quickly dissected on dry ice after the animal are sacrificed. Based on the changes measured in LCBF, we defined an imaginary line, which is parallel to the superior sagittal sinus (SSS) and 3.0 mm lateral to the bregma, to divide the superior limb of the penumbral (left side to the line) and the ischemic core (right side to the line) cortices in the ischemic brain. Cortical dissection is continued between the rhinal fissure and the lateral olfactory tract. The inferior margin of ischemic core was defined as the cortical brain tissues located superiorly to the level of the rhinal fissure. In in vitro model, cultured neurons were harvested at indicated time after ischemic stroke. Samples were homogenized in lysis buffer, containing 1% Triton X-100, 20 mM Tris-HCl (pH7.5), 150 mM NaCl, 0.5% deoxycholate, 1 mM EDTA, 0.1% SDS, and is centrifuged at 18,000 g for 60 mins at 4°C. Protein concentrations of each sample were determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). Cell lysates of 25 μg protein were separate by 10% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE), and was transferred onto polyvinylidene difluoride (PVDF) microporous membranes (IPVH00010, Millipore, Billerica, MA, USA). Membranes were blocked with 5% milk, then probed with primary antibodies against BDNF (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), GAP-43, PSD-95 (1:10,000 and 1:1,000; Chemicon International, Temecula, CA, USA), and SNAP-25 (1:10,000; Serotec, Raleigh,NC, USA) and finally incubated with horseradish eroxidase–conjugated
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immunoglobulin (1:5,000; Chemicon International). Bound antibody was visualized with the Amersham ECL system (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). Membranes are then probed for actin (1:10,000; Chemicon International). Optical densities were measured by a Luminescent Image Analyzer (Fujifilm LAS-3000; Fuji Photo Film Co., Tokyo, Japan).
Immunohistochemistry of BDNF, GAP-43, PSD-95, and SNAP-25
Coverslips cultured with mixed neuronal-glial cells were pretreated with melatonin before exposed to glutamate (300 μM). The coverslips were processed with primary antibodies against BDNF (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), GAP-43, PSD-95 (1:10,000 and 1:1,000; Chemicon International, Temecula, CA, USA), and SNAP-25 (1:10,000; Serotec, Raleigh,NC, USA) at 4°C overnight. Appropriate secondary antibody conjugated with biotin (1:100, Jackson ImmunoResearch, Inc., West Grove, PA, USA) will then be added, followed by the FITC-conjugated streptavidin and Texas red-conjugated streptavidin (1:100, Jackson ImmunoResearch, Inc.). Sections were co-incubated with
4’,-6-Diamidino-2-phenylindole (DAPI) for 30 secs. Negative controls with preimmune serum will also be included, and no immunoreactivity were detected in each protocol.
Fluoro-Jade staining
Fluoro-Jade is an anionic tribasic fluorescein derivative. Neuron degenerating processes were detected with Fluoro-Jade (Histochem, Jefferson, AK) as originally described by Schmued et al. The tissue was fully dry and incubated in each of the following solutions for the time indicated: 70% alcohol, 2 min; distilled water (dH2O), 2 min; 0.06% potassium permanganate (KMnO4), 10 min; dH2O, 2 min; 0.0001% Fluoro-Jade in 0.1% acetic acid, 10 min; dH2O, 1 min. Stained sections were allowed to dry at 50℃. Dried slides were cleared in xylene and coverslips with few drops of mounting medium. The number of Fluoro-Jade positive cells were counted in six non-overlapping regions (500×400 μm2) sampled at ischemic core (three in cortex and three in striatum). Cell counts were expressed as the number of Fluoro-Jade positive cells per mm2 for cortical and striatal region.
Statistical analysis
Data analyses were conducted by an investigator unaware of treatment protocols. Neurobehavioral scores were expressed as the median ± 95% confidence interval (CI) and were analyzed by Mann-Whitney U test. The other data were examined for normality to ensure normal/approximately normal distribution, and were
expressed as the mean ± standard deviation (S. D.). Paired Students’ t test was used to evaluate the response to a change in conditions, and unpaired Students’ t test were used to evaluate differences between groups. P < 0.05 were selected for statistical significance.
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Results
The vulnerability in young and aged mice to MCA ligation
In our study, MCA ligation's mortality rates in young and aged mice were 10.2% and 41.4%, respectively. We conducted an animal model to test whether melatonin has discriminated- neuroplastic effect in aged mice. We first confirmed the levels of cellular senescence by cytochemical staining of lipofuscin and senescence associated b-galactosidase. The number of lipofuscin-load cells in aged mice's cortex was higher than the young mice. Both young and aged mice subjected to permanent MCA occlusion did not exhibit spontaneous hyperthermia but rather displayed modest hypothermia. The postischemic core temperature and LCBF were not statistically different at each sampling time interval between study groups.
The effects of melatonin to the brain subjected to MCA occlusion
Relative to controls, both young and aged mice treated with melatonin had significantly reduced brain infarction- volume and size. Melatonin treatment (Young-melatonin -injected group n=8; Aged-melatonin -injected group n=7) significantly reduced infarction volume, and reduced individual cortical lesion size, and significantly increased the number of surviving neurons, compared with control data (Young-vehicle-injected group n=8; Aged-vehicle-injected group n=8) in both young and aged mice. Additionally, both young and aged mice treated with melatonin increased the numbers of the surviving neurons in the penumbral cortex. These results indicated that treatment with melatonin reduced gray matter damage in young and aged mice subjected to MCA occlusion.
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Six random and nonoverlapping (500 X 400 μm) regions in the borders of the ischemic parietal cortex and striatum were selected for counting surviving neurons. Scale bar = 5 mm. Percentage infarction volume (E), individual cortical lesion size (F), and the number of surviving neurons (G) are expressed as a percentage of the contralateral (control) hemisphere. Data are represented as the mean ± S.D. * P < 0.05 by students-t test.
Treatment with melatonin (5 mg/kg) before middle cerebral artery (MCA) occlusion, reduced gray matter damage at 24 hours after permanent MCA occlusion in both young and aged mice. The cresyl violet-stained coronal sections were from representative animals from Young-vehicle-injected group (A); Young-melatonin- injected group (B); Aged-vehicle-injected group (C); Aged-melatonin-injected group (D).
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We then conducted neurological behavioral test at 24 h after MCA ligation. As shown in table, melatonin improved the sensorimotor behavioral scores in young and aged mice subjected to MCAo. Treatment with melatonin (5 mg/kg) before middle cerebral artery (MCA) occlusion improved sensorimotor behavioral scores at 24 hours after permanent MCA occlusion.
Neurobehavioral test
Weight data and neurological behavioral scores are expressed by mean ± S.D. and by median (95% CI), respectively. Melatonin (5 mg/kg) treatment improved sensorimotor neurological scores compared with vehicle-injected control data in both young and aged a mice. , P < 0.05 Young -melatonin-injected data versus young-vehicle-injected control b data; P< 0.05 Aged-melatonin-injected data versus aged-vehicle-injected control data;c d, P < 0.05 Young-vehicle-injected control data versus aged-vehicle-injected control data ; , P < 0.05 Young-melatonin -injected data versus aged-melatonin -injected data, respectively by students-t test. Rotarod
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behaviors at 24 hours after permanent MCA occlusion in both young and aged mice. Permanent MCAo reduced locomotor behaviors in young and aged mice compared with sham-operated data (Young-sham group n=3; Aged-sham group n=3; Young-vehicle-injected group n=23; Aged-vehicle-injected group n=18) assessed by accelerated and fixed modes of rotarod motor performance test. Melatonin (5 mg/kg)-treated animals (Young-melatonin-injected group n=24; Aged-vehicle-injected group n=17) had improved locomotor behaviors compared with vehicle-injected control data in both young and aged mice. Data are represented as the mean ± S.D. * P < 0.05 by students-t test.
We then conduct rotarod experiment test to verify the sensory and motor function for each group. The results have been shown at the above figure. In the sham-operated group, both the fix and accelerated modes of rotarod motor performance in aged mice were worse than that of young mice. Both groups of young and age mice subjected to MCAo followed by treatment with melatonin showed better performance in the test of rotarod than vehicle-injected animals in tests either in fixed or accelerated modes.
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Lipofuscin-Age marker
Lipofuscin accumulation in aged mice.
Epifluorescence photomicrograph of representative lipofuscin-loaded cells from layer III-IV of contralateral (intact) hemisphere in the 3-month (young) group (n=8) (A) and 12-month (aged) group (n=8) (B).
Lipofuscin-loaded cells (%) in the contralateral cortex of aged mice is much higher than young mice (D). Data are represented as the mean ± S.D. * P < 0.05 ; ** P < 0.01 by students-t test. Scale bar= 50 μm; =10 μm in the magnification inset.
Lipofuscin (A) and senescence-associated-galactosidase (B) accumulation in primary cultured
neurons. Lipofuscin-load cells (%) and -galactosidase (%) in the day 15-cultured neuron is higher than day 10-cultured neuron.
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Treatment with melatonin (5 mg/kg) before middle cerebral artery (MCA) occlusion resulted in better dendritic arborization at 24 hours after permanent MCA occlusion in both young mice and aged mice. Data are represented as the mean ± S.D. * P < 0.05 by students-t test.
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Melatonin have higher dendritic spine density and better dendritic arborization both in young and aged mice subjected to middle cerebral artery (MCA) occlusion.
Vehicle-injected groups (Young-vehicle-injected group n=8; Aged-vehicle-injected group n=8) had a decreased dendritic spine density in the layer II-III, layer III-IV of the penumbra, layer V-VI of the ipsilateral (ischemic) cortex, and layer V-VI of contralateral (intact) cortex compared to sham-operated controls
(Young-sham group n=3; Aged-sham group n=3). Melatonin (5 mg/kg) treatment (Young-melatonin-injected group n=8; Aged-melatonin-injected group n=8) had significantly led to higher dendritic spine density of the second- and third-order dendrites at the layer II-III, layer III-IV and V-VI of the ipsilateral cortex and layer V-VI of the contralateral cortex, relative to vehicle-injected controls in both young and aged mice.
Furthermore, vehicle-injected groups (Young-vehicle -injected group n=8; Aged-vehicle-injected group n=8) had a decreased total dendritic branches and total dendritic length in the layer II-III, layer III-IV of the penumbra and layer V-VI of the ipsilateral (ischemic) cortex and layer V-VI of contralateral (intact) cortex compared to sham-operated controls (Young-sham group n=3; Aged-sham group n=3). Melatonin (5 mg/kg) treatment (Young-melatonin-injected group n=8; Aged- melatonin-injected group n=8) had significantly elevated total dendritic branches and total dendritic length relative to vehicle-injected controls of the pyramidal neurons in layer II-III, layer III-IV of the penumbra of the ipsilateral cortex.
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Treatment with melatonin (5 mg/kg) before middle cerebral artery (MCA) occlusion decreased neuronal degeneration at 24hrs after permanent MCA occlusion in both young and aged mice. Photomicrographs of representative degenerating neurons from penumbra layer III-IV of ipsilateral (ischemic) hemisphere of Young- vehicle-injected group (A); Young-melatonin-injected group (B); Aged-vehicle-injected group (C) and Aged-melatonin -injected group (D). The numbers of degenerating neurons in the ipsilateral parietal cortex (E). Scale bar=50 μm; =10 μm in the magnification inset. Data are represented as the mean ± S.D. * P < 0.05 by students-t test.
Treatment with melatonin (5 mg/kg) before middle cerebral artery (MCA) occlusion resulted in higher dendritic spine density at 24 hours after permanent MCA occlusion in both young mice and aged mice. The phtomicrographs of representative dendritic segments from pyramidal neurons sampled at dufferent layer of contralateral (intact) hemisphere of young and aged mice. Data are represented as the mean ± S.D. * P < 0.05 by students-t test.
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Sholl analysis
Treatment with melatonin (5 mg/kg) results in dendritic development in young and old MCAo mice Vehicle-injected groups (Young-vehicle -injected group n=8; Aged-vehicle-injected group n=8) had a
decreased the number of branch segment per branch order in the layer II-III, layer III-IV of the penumbra and layer V-VI of ipsilateral (ischemic) hemisphere compared to sham-operated controls (Young-sham group n=3; Aged-sham group n=3) (A,B,C). Melatonin (5mg/kg) treatment (Young-melatonin-injected group n=8;
Aged-melatonin -injected group n=8) had a significantly resulted in greater number of branch segments per branch in the layer III-IV of the penumbra of ipsilateral cortex. Data are represented as the mean ± S.D. * P < 0.05 by students-t test.
Treatment with melatonin (5 mg/kg) elevated the number of dendrite -sphere intersection in both young mice and aged mice.
The left panel indicated the sketch map of sholl concentric sphere analysis. Vehicle-injected groups (Young-vehicle-injected group n=8; Aged-vehicle-injected group n=8) had a decreased the number of dendrite-sphere intersections at increasing distances from the cell body in the layer II-III, layer III-IV of the penumbra and layer V-VI of ipsilateral (ischemic) hemisphere compared to sham-operated groups (Young-sham group n=3; Aged-sham group n=3) (A,B,C). Melatonin (5mg/kg) treatment (Young-melatonin-injected group n=8; Aged-melatonin -injected group n=8) significantly elevated number of dendrite-sphere intersections at increasing in the layer II-III and layer III-IV of the penumbra of ipsilateral cortex (A,B). Data are represented as the mean ± S.D. * P < 0.05 by students-t test. Due to the limitation of file size, the figures have been skipped at current report.
Melatonin increased the expression of neuroplasticity-associated protein in aged-cultured neurons In order to establish a link between senescence and culture time, cortex cultured neurons were culture until 20 days. The levels of lipofuscin and senescence-associated b-galactosidase were detected every 5 days. The proportion of the senescent cells steadily increased in neuron cultures. Consistent with in vivo aged model about 40% of cells stained lipofuscin and senescence-associated b-galactosidase positive at 20 days in vitro.
Therefore, experiments were undertaken on cultured neurons at 20 days in vitro. Our results found that
melatonin treatment significantly increased BDNF, GAP-43, and PSD-95 but not SNAP-25 protein expression in young-cultured neurons without exposed OGD excitotoxicity. Additionally, melatonin treatment also
increased BDNF, GAP-43, and PSD-95 protein expression in aged-cultured neurons without exposed OGD excitotoxicity. Additionally, melatonin treatment also increased BDNF, GAP-43, and PSD-95 protein expression in aged-cultured neurons without exposed OGD excitotoxicity. Furthermore, we investigated the neuroplasticity effect of melatonin in aged-cultured neurons exposed OGD excitotoxicity. We found that melatonin (20 µM) improved the expressions of the GAP-43 by 1.41 and 1.38 folds, compared with controls at 24 and 48 hr, respectively. Melatonin (50 µM) improved the expressions of the GAP-43 by 1.49, compared with controls at 24 hr. Melatonin 20 µM and 50µM also significantly improved the expressions of PSD-95 by 1.32
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and 1.21 folds in age-culture neurons at 24 hr postischemic. However, melatonin treatment did not affect the SNAP-25 levels in aged-cultured neurons.
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Melatonin increased BDNF protein expression in young and aged mice after MCA occlusion. Pretreatment with melatonin increased the intensity of BDNF in ischemic core of young (A) and aged (B)mice as detected by immunofluorescence assay compared to control.
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Melatonin increased neuroplasticity-associated protein expression in young and aged mice after MCA
occlusion. Pretreatment with melatonin increased the intensity of GAP-43 (A and B) and SNAP-25 (C and D) in ischemic core of young and aged mice as detected by immunofluorescence assay compared to control.
Melatonin increased the expression of neuroplasticity-associated protein in vivo after insult.
Relative to controls, melatonin-treated stroke animals also caused a significant improvement in GAP-43, SNAP-25, and PSD-95 expressions as well as the BDNF in the ischemic brain in young- and age-mice.
Melatonin-treated young and aged had a significantly increased protein levels of the BDNF at the ischemic core. The age-cultured neuron treated with 20 µM and 50 µM melatonin improved the expressions of the BDNF proteins by 1.35 and 1.18 folds, compared with controls at 24 hr, respectively. Additionally, melatonin (20 µM) improved the expressions of the GAP-43 by 1.41 and 1.38 folds, compared with controls at 24 and 48 hr, respectively.
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Melatonin increased neuroplasticity-associated protein expression in young and aged mice after MCA
occlusion. Pretreatment with melatonin increased the intensity of GAP-43 (A and B) and SNAP-25 (C and D) in ischemic core of young and aged mice as detected by immunofluorescence assay compared to control.
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Melatonin increased neuroplasticity-associated protein expression in young and aged mice after MCA
occlusion. Pretreatment with melatonin increased the intensity of GAP-43 (A and B) and SNAP-25 (C and D) in ischemic core of young and aged mice as detected by immunofluorescence assay compared to control.
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We conducted in vitro study to test if melatonin induces dendritic development during the basal condition. In the above figure, the total dendritic lengths increased in the presence of melatonin with a concentration of 20, 50, and 100 mM at least from 24 h to 72 h. Our data indicated that melatonin exhibit the effect of neuroplasticity without the precondition of brain insults.
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Melatonin increased neuroplasticity associated protein expression in aged-cultured neurons. BDNF, GAP-43, PSD-95 and SNAP-25 in aged-cultured neurons. Melatonin (20 and 50 µM) improved the expressions of BDNF, GAP-43, PSD-95 and SNAP-25, compared with vehicle-treated controls.
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A B
C D
Melatonin increased neuroplasticity associated protein expression in aged-cultured neurons. BDNF, GAP-43, PSD-95 and SNAP-25 in aged-cultured neurons. Melatonin (20 and 50 µM) improved the expressions of BDNF, GAP-43, PSD-95 and SNAP-25, compared with vehicle-treated controls.
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A B
C
Melatonin increased neuroplasticity associated protein expression in young mice after MCA occlusion. Western blotting analysis for BDNF, GAP-43, PSD-95, and SNAP-25 in the ischemic brain. The photographs show typical pattern of changes of BDNF (A), GAP-43 (B), PSD-95 (C), and SNAP-25 (D) protein
expressions in the contralateral intact (L) and the ischemic core (R) areas at 24 hrs postinsult. Melatonin increased BDNF, GAP-43, PSD-95, and SNAP-25 protein levels in ischemic core in young mice, compared with controls.
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Conclusion
In summary, we demonstrated that administration with melatonin induces neuroplasticity in young and age stroke models in vivo and in vitro. Our results showed that melatonin protects neurons against ischemic injury by beneficial neuroplasticity, which improved neural function recovery. Melatonin treatment might render the increases of dendritic spine density and dendritic arborization in the ischemic hemisphere and increase the rise of dendritic spine density in the non-ischemic hemisphere in young and aged mice. In comparing the potency in melatonin regarding young and old mice, the medicine exerts a similar improvement in mice subjected to MCAo insults. Furthermore, treatment with melatonin reduces
ischemia-induced neuronal damage and increasing BDNF, GAP-43, and PSD-95 protein expressions, and, thus, improves the locomotor and neurobehavioral outcomes at postischemic. Considering the wealth of information on the beneficial effects of melatonin in experimental stroke, it may be worth further investigating in humans' field of ischemic stroke.
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科技部補助專題研究計畫成果報告自評表
請就研究內容與原計畫相符程度、達成預期目標情況、研究成果之學術或應用價 值(簡要敘述成果所 代表之意義、價值、影響或進一步發展之可能性)、是否適 合在學術期刊發表或申請專利、主要發現(簡 要敘述成果是否有嚴重損及公共利 益之發現)或其他有關價值等，作一綜合評估。 1. 請就研究內容與原計畫相符程度、達成預期目標情況作一綜合評估 ■達成目標 □ 未達成目標(請說明，以 100 字為限) □ 實驗失敗 □ 因故實驗中斷 □ 其他原因 說明: 2. 研究成果在學術期刊發表或申請專利等情形: 論文:□已發表 □未發表之文稿 ■撰寫中 □無 專利:□已獲得 □申請中 ■無 技轉:□已技轉 □洽談中 ■無 其他:(以 100 字為限) 3. 請依學術成就、技術創新、社會影響等方面，評估研究成果之學術或應用價值(簡要敘述成果所代 表之意義、價值、影響或進一步發展之可能性)，如有嚴重損及公共利益之發現，請簡述可能損及 之相關程度(以 500 字為限) 本研究利用中風動物模型，探討褪黑激素對於年輕老鼠與老老鼠的效能差異，以及作用機轉差異 性，並探討非中風情況下，褪黑激素對神經細胞的效能，並探究其機轉。我們發現動物正常老化 下，其神經功能會變差，受中風之後其嚴重程度會較大，並有較高的死亡率。然而在存活下來的 動物身上，我們發現褪黑激素對動物感覺運動功能改善程度不亞於年輕的老鼠。此外，對於正常 的神經細胞，給予褪黑激素會促進神經分支的形成，使神經結構更豐富。此結果暗示正常人服用 褪黑激素也會促進神經重塑作用。在重塑的機制上，我們發現褪黑激素會造成 BDNF 的表現上升， 之後帶動 GAP43, SNAP-25, PSD-95。由於過去曾經有研究報告顯示褪黑激素所造成的神經保護作 用與 M1,M2 接受器無關。我們進一部探討褪黑激素在神經重塑作用的上游機制。我們發現利用 M1,M2 受器的拮抗劑與刺激劑會對 BDNF 的表現造成抑制與誘發的作用。由於 BDNF 是 GAP43, SNAP-25, PSD-95 等訊息傳遞因子的上游，因此我們傾向認為褪黑激素是透過 M1,M2 受器的路徑 來造成神經重塑作用。此結果與褪黑激素在神經保護作用上不同。總結而言，褪黑激素可能透過 不同的路徑，來促進神經保護與神經重塑的效果。32 108 年度專題研究計畫成果彙整表 計畫主持人: 計畫編號: 107-2314-B-006-027-MY2 計畫名稱: 利用年老小鼠中風模式探討褪黑激素結合agomelatine於中風後神經保護與神經重塑機制上 扮演的角色 (II-III) 成果項目 量 化 單 位 質化 (說明:各成果項目請附佐證資料或細 項說明，如期刊名稱、年份、卷期、起訖 頁數、證號...等) 國 內 學術性論文 期刊論文 0 篇 研討會論文 0 專書 0 本 專書論文 0 章 技術報告 0 篇 其他 0 篇 國 外 學術性論文 期刊論文 2 篇 撰稿中 研討會論文 0 專書 0 本 專書論文 0 章 技術報告 0 篇 其他 0 篇 參 與 計 畫 人 力 本國籍 大專生 0 人 次 碩士生 0 博士生 0 博士級研究人員 0 專任人員 1 非本國籍 大專生 0 碩士生 0 博士生 0 博士級研究人員 0 專任人員 0 其他成果 (無法以量化表達之成果如辦理學術活動、獲得獎項、 重要國際合作、研究成果國際影響力及其他協助產業 技術發展之具體效益事項等，請以文字敘述填列。)
