Administrative Information

المعلومات الادارية

 

عنوان المشروع-  Project Title

بلغة اجنبية:

Mechanisms of neural injury caused by chronic hypoxia in the developing brain

باللغة العربية:

كيفيّة اصابة الخلايا الدماغية الناتجة عن نقص الأوكسيجين المزمن عند الولادة

 

الباحث الرئيسي Principal Investigator -

الاسم

 Name

المؤسسة

Institution

الوظيفية

Post

العنوان

Address

العنوان الالكتروني

e-mail

رقم الهاتف

Telephone

Mohamad Mikati

AUB

Head of department

P.O.Box 11-0236 Riad el Solh-Beirut 1107 2020

mamikati@aub.edu.lb

01 374374

الباحثون المشاركون Co-Workers -

None

المدة التعاقدية للمشروع Duration -: ثلاث سنوات (3 years)

 

Scientific Information

المعلومات العلميّة

الهدف-  Objectives

1-Determine if there is evidence of Programmed Cell Death (PCD) in the developing brain subjected to acute hypoxia 3 and 7 days after the insult.

2-Find out if there is an increase in erythropoietin during chronic hypoxia which could suggest a neuroprotective role of this peptide during chronic hypoxia.

3-Find out if the administration of exogenous erythropoietin has neuroprotective effects in the acute hypoxia model.

4/5- Find out the effects of topiramate on the long-term sequelae of chronic, acute, and acute with chronic hypoxia (Handling, Open field, Water maze tests, and routine histologic methods) and determine if topiramte can prevent the deleterious effects of acute hypoxia when given just before or just after the hypoxic insult.

 

ألانجازات المحققة   Achievements -

P10 rats were divided into 2 groups: Control group sham manipulated and Hypoxia group subjected to acute hypoxia. Rats were sacrificed at 1 day (n=10/group), 3 days (n=18/group), 7 days (n=12/group) post hypoxic insult. TUNEL scoring was performed blindly according to a scale from 0 to 4 with 4 being the most severe. The TUNEL scores (apoptosis scores) did not differ between the 2 groups (p>0.05 in all comparisons). Mean±SE of total TUNEL scores (combined data of all hippocampal subfields) were: P11: 0.00±0.00, 0.3±0.15; P13: 0.00±0.00, 0.27±0.11; P17: 0.00±0.00, 0.25±0.13 for the Control and Hypoxia groups respectively.  

 

   We studied the erythropoietin level in the serum of control, acute, chronic and chronic with superimposed acute hypoxia treated rats N=35 rats. Erythropoietin levels in the ctrl and ac rats were not different (118.86 pg/ml ± 6.62 and 209.38 pg/ml ± 13.99 respectively, P = 0.8). The ch+ac hypoxia group had significantly higher levels as compared to the ch group (6803.021 pg/ml ± 559.97 and 2983.90 pg/ml ± 694.49 respectively, P < 0.000001), and both were higher than the ctrl and ac groups (P < 0.000046).  

 

   Adult Sprague-Dawley rats received single intraperitoneal high-dose injections of recombinant Epo (1unit/g). Plasma and cerebrospinal fluid (CSF) samples were collected sequentially between 30 and 1260 minutes after the injection and Epo was measured. Erythropoietin was detected in the plasma (222.09 mlUnits/ml) 30 minutes after the injection, peaked to 4547.93 mlUnits/ml at 5 hours, and decreased to 645 mlUnits/ml after 21 hours, with a half life of 7.5 hours. CSF Epo concentrations paralleled the rise in plasma levels and peaked to11 mlUnits/ml, 5 hours after the injection.

 

   Also, to determine if Erythropoietin (EPO) has protective effects against subsequent seizure susceptibility and seizure related neuronal injury in rat pups subjected to acute hypoxia at P10. Four groups of rats were manipulated at P10, as described below, then all received kainic acid (KA) (10mg/kg i.p.) at P29:  Hypoxia-NS-KA group (n=11): subjected to acute hypoxia (down to 4% O2), and then immediately received saline i.p. Hypoxia-EPO-KA group (n=10): subjected to acute hypoxia and then immediately received EPO (1000U/Kg) i.p. Normoxia-NS-KA group (n=11): sham manipulated and injected with saline. Normoxia-EPO-KA group (n=10): sham manipulated then immediately injected with EPO (1000U/Kg) i.p. After receiving KA at P29, all rats were monitored using videotape techniques, and were sacrificed at P31. TUNEL and Hoechst stains to assess for apoptosis, and regular histology for hippocampal cell counts were performed. We found that administration of the single dose of Erythropoietin directly after an acute hypoxic event at P10 resulted at P29 in increased latency to Forelimb clonus seizures, reduced duration of these seizures, protection against hippocampal cell loss, and decreased hippocampal apoptosis in the Hypoxia-EPO-KA group as compared to the Hypoxia-NS-KA group. We concluded that these data support the presence of favorable protective effects of Erythropoietin against the long term consequences of acute hypoxia in the developing brain and raise the possibility of its investigation as a potential neuroprotective agent after human neonatal hypoxic encephalopathy.

 

    To determine whether topiramate has long-term neuroprotective effects in pups subjected to chronic, acute, or to chronic with superimposed acute hypoxia. Pups were subjected to chronic (10% O2 from P0-P21) or to acute (down to 4% O2 on P10) hypoxia and received a daily intraperitoneal (i.p.) injections of topiramate (TPM) 30mg/kg from P0 till P21 (Ch+TPM and Ac+TPM groups). Two other groups, acute and chronic were treated similarly and injected with PBS, the vehicle of TPM (Ch+V and Ac+TPM groups. Controls (21% O2) were daily injected from P0 till P21 with PBS (Ctrl+V group). The pups were weighed before every injection. Behavioral tests (handling, Open field, Morris water maze and Probe tests) were performed starting P81. On P98 rats were sacrificed and their brains were dissected for histological studies. We found that the survival of the Ch+V, Ch+TPM, Ac+V, Ac+TPM and Ctrl+V groups was 75%, 0%, 87.5%, 87.5% and 50% respectively. The handling test showed increase aggressivity in the Ac+V group as compared to the controls and to Ac+ TMX group  (p=0.0050 and p=0.0034 respectively). In addition, Ac+TMX group was not different from the control group p=0.733. Morris water maze test showed that Ac+V group had long term memory impairment as compared to Ac+TMX group p=0.0078. Ac+V and Ac+TMX groups were not different from the control group (p=0.23 and p=0.27 respectively). We concluded that topiramate has favorable effect in preventing the consequences of perinatal hypoxia.

 

آفاق البحث  Perspectives -

غير متوفر

 

المنشورات والمساهمات في المؤتمرات-  Publications & Communications

1-     Mikati MA, Zeinieh MP, Kurdi RM, Harb SA, El Hokayem JA, Daderian RH, Shamseddine A, Obeid M, Bitar FF, El Sabban M, Long term effects of acute and of chronic hypoxia on behavior and on hippocampal histology in the developing brain, Brain Research Developmental Brain Research, 2005(157): 98- 102

2-     Mikati MA, El Hokayem JA, El Sabban M, Effect of a single dose of erythropoietin on subsequent seizure susceptibility in rats exposed to acute hypoxia at P10, Epilepsia, 2007(48): 157-81

3-     Abstracts published in Epilepsia Vol. 46, Suppl. 6, 2005 poster numbers p593 and p1182.

 

 

 

موجز باللغة العربية عن نتائج البحث Abstract in Arabic

إن البحث الذي قمنا به في هذا الإقتراح تناول نقاطاً عدة و هنا ملخص عن بعض النتائج التي توصلنا اليها.

          النقطة الأولى تناولت البحث عمّا اذا كان نقص الأوكسيجين الحاد لدى  الجرذان الذين يبلغ عمرهم عشرة أيام يؤدي الى موت الخلايا الدماغية المبرمج و ذلك بعد يوم، ثلاثة أيام، و سبعة أيام بعد الإصابة. و قد برهن صباغ الTUNEL أنه ليس هناك موت للخلايا في الأوقات التي تمّت دراستها بعد الإصابة.

النقطة الثانية تناولت البحث عمّا إذا نقص الأوكسيجين الحاد، أو نقص الأوكسيجين المزمن، أو نقص الأوكسيجين المزمن المرفق بنقص الأوكسيجين الحاد، يؤدون الى ارتفاع بمعدل الإريثروبوتين (Erythropoietin) في الدم و الذي يمكن أن يؤمن الحماية ضد الإصابة. و برهنت الدراسات التي قمنا بها أن معدل الEpo ارتفع فوق المعدل العام فقط في حالتي نقص الأوكسيجين المزمن و نقص الأوكسيجين المزمن المرفق بنقص الأوكسيجين الحاد.

          النقطة الثالثة تناولت البحث عمّا إذا حقن جرعة واحدة من الEpo قد يؤدي الى الحماية ضد الإصابة جرّاء نقص الأوكسيجين. و قد تمّ ارتفاع معدل الEpo  بشكلٍ ملحوظ بدءاً من نصف ساعة بعد الحقنة و وصل الى أعلى ارتفاع بعد خمس ساعات من الحقنة و هذا الارتفاع رافقه ارتفاع في معدل الEpo في سائل النخاع الشوكي. كما أننا بحثنا عمّا إذا كان لل  Epo دور في التخفيف من النوبات العصبية و ما تؤدي اليه بعد نقص الأوكسيجين الحاد لدى الجرذان البالغين من العمر ۱۰ أيام و ذلك من خلال إثارة هذه النوبات بحقنة من ال kainic acid في عمر التاسع و العشرين يوم. إن حقنة الEpo فوراً بعد الإصابة بنقص الأوكسيجين الحاد أدّت الى التخفيف من حدة النوبات العصبية و تخفيض موت الخلايا و موت الخلايا الدماغية المبرمج.

          أما النقطة الرابعة فتناولت البحث عن دور دواء ال Topiramate في تخفيف تأثير نقص الأوكسيجين الحاد، أو نقص الأوكسيجين المزمن، أو نقص الأوكسيجين المزمن المرفق بنقص الأوكسيجين الحاد على المدى الطويل  و ذلك من خلال حقنة يومية من هذا الدواء منذ الولادة و حتى اليوم الواحد و العشرين. برهنت الدراسات أن الTopiramate ساعد على التخفيض من مخاطر نقص الأوكسيجين الحاد من خلال التخفيف من درجة الإستثارة الهجومية   و تحسين الذاكرة لدى المصابين.