1. Right Inferior Cortex, HIE/NT/hASC Left Inferior Cortex, HIE/NT/hASC
Effect of Hypothermia Therapy and Human Adipose-derived Stem Cells (hASCs) on Brain Cortex in Neonatal Rats post Hypoxic Ischemic Encephalopathy
Blanca Iriarte MD1, Melissa February, MD2, Nikolas Kappy, MD3,
Shaohua Chang PhD3, Russel Buono PhD4, Alla Kushnir MD5
Mercy Health System, Philadelphia, PA; 2. Children's Hospital of Michigan, Detroit, MI;
3. Cooper University Hospital, Department of Surgery, Camden, NJ; 4. Cooper Medical School of Rawan University, Camden, NJ; 5. Cooper University Hospital, Department of Pediatrics, Camden, NJ.
Abstract
BACKGROUND: Hypoxic ischemic encephalopathy (HIE) continues to be a major cause of morbidity and mortality in the neonatal population. Currently, therapeutic hypothermia is the only therapy proven to be neuroprotective post HIE. Bone marrow-derived mesenchymal stem cells (BMMSCs) have been shown to be therapeutic in animal models of HIE.  Human derived adipose stem cells (hASCs) share all the properties of BMMSCs; however, they are more easily acquired and suffer fewer negative effects.
OBJECTIVE: To determine whether hypothermia and hASC have additive neuroprotective effects in neonatal rats post HIE by comparing brain cortex between treatments.
METHODS: Seven-day-old Sprague Dawley (SD) rats were subjected to left carotid artery ligation or isolation alone as a sham control. After 2 hours of recuperation the ligation group was subjected to hypoxia with 8% O2/92% N2 for 120 minutes. After that, rats were either kept normothermic (NT) or hypothermic (HT) for 4 hours. Forty-eight hours after surgery half of the rats received hASCs and half normal saline (NS) via tail vein. The brain tissues were paraffin embedded, sectioned and stained for Nissl bodies using Cresyl Violet. Thickness of cortical plate in upper (LU and RU) and lower (LL and RL) quadrants of coronal sections was measured and compared between the 8 groups using microscopy and image analysis.
RESULTS: There was no significant difference in cortical thickness between groups in the superior quadrants. There was no significant difference in superior quadrants in normothermia (NT) and hypothermia (HT) groups (p=0.22 and 0.26 respectively). Cortical thickness was significantly different in the inferior region between all samples (p=0.05) and in the normothermia only sub-group (p=0.006). In post-hoc analysis, there was statistical cortical atrophy noted in HIE/NT/NS compared to those who had HIE and were exposed to hypothermia (p<0.05). Cortical thickness was similar in shams to those post HIE with hASC.
CONCLUSIONS: There was significant cortical sparing with potential neuroprotective effect in normothermic animals post HIE who received hASC injection. This protection was not seen in animals treated with hypothermia. This is most likely due to the neuroprotective effect of hypothermia alone, which was not attenuated by addition of hASC.
Methods
IRB and the institutional animal care and use committee approved.
Adipose tissue was obtained by liposuction of subcutaneous peri-umbilical fat from cosmetic surgery patients.
Stromal vascular fraction (stem cell layer) collected.
Adherent hASC expanded over days with refeeding every 3rd day, (yield 108 cells).
On day 7 of life left common carotid artery was ligated or isolated.
Hypoxia induced using 8% oxygen and 92% nitrogen for 120 minutes.
Half of all pups underwent therapeutic hypothermia (HT) at 32º C and half maintained in 37º C for 4 hours.
Body temperature was monitored every10 min for 30 min, then every 30 minutes.
Transplantation of hASC to half or normal saline (NS) to the other half, after 48 hours via IV using tail vein.
The brain tissues were paraffin embedded, sectioned and stained for Nissl bodies using Cresyl Violet.
Thickness of cortical plate in left and right upper (LU and RU) and lower (LL and RL) quadrants of coronal sections was measured and compared between the 8 groups using microscopy and image J analysis.
Multiple ANOVA with Dunnet post-hoc comparison was used to analyze the data.
Right Inferior Cortex, HIE/NT/hASC
Left Inferior Cortex, HIE/NT/hASC
Introduction
Hypoxic ischemic encephalopathy (HIE) continues to be a major cause of morbidity and mortality in the neonatal population. Currently, therapeutic hypothermia is the only therapy proven to be neuroprotective post HIE. Bone marrow-derived mesenchymal stem cells (BMMSCs) have been shown to be therapeutic in animal models of HIE.  Human derived adipose stem cells (hASCs) share all the properties of BMMSCs; however, they are more easily acquired and suffer fewer negative effects.
Comparing of brain cortex thickness is one mode of determining whether there is neuroprotective effects in neonatal rats post HIE. *
Demographics
Results
Rate of surgical survival was 87 % (104/120).
There was no significant difference in cortical thickness between groups in the superior quadrants.
There was no significant difference in superior quadrants in normothermic (NT) and hypothermic (HT) groups (p=0.22 and 0.26 respectively).
Cortical thickness was significantly different in the inferior region between all samples (p=0.05) and in the normothermic only sub-group (p=0.006).
In post-hoc analysis, there was statistical cortical atrophy noted in HIE/NT/NS compared to those who had HIE and were exposed to hypothermia (p<0.05).
Cortical thickness was similar in shams to those post HIE who were treated with hASC.
Conclusions
There was significant cortical sparing with potential neuroprotective effect in normothermic animals post HIE who received hASC injection. This protection was not seen in animals treated with hypothermia. This is most likely due to the neuroprotective effect of hypothermia alone, which was not attenuated by addition of hASC.
Sham Saline
Sham Cells
Ligation/HT Saline
Ligation/HT Cells
Ligation/NT Saline
Ligation/NT Cells
Male gender (%)
48%
50%
62%
58%
53%
67%
Birth Weight (g)
14.1 ± 1.4
14.9 ± 1.7
13.2 ± 2.1
13.4 ± 1.1
14.4 ± 1.6
13.6 ± 1.3
Weight Day 14
25.9± 5.2
28.2 ± 4.5
29.3±5.1
27.7 ± 4.6
27.7±7
29.4 ± 4.8
Weight Day 28
79.9 ± 13.5
87.8 ± 14.3
77.5 ± 13.6
74.1 ± 13.6
76 ± 12.1
73 ± 17.6
Weight Day 42
175.6 ± 27.2
177.9 ± 33.9
165 ± 21.3
163 ± 29.5
165.1 ± 22.1
170.2 ±33.5
References
1. Vannucci RC, Perlman JM. Interventions for Perinatal Hypoxic-Ischemic Encephalopathy Pediatr 1997
2. Allan WC. The clinical spectrum and prediction of outcome in hypoxic-ischemic encephalopathy. Neoreviews 2002; 3; e108-e115
3.Delivoria-Papadopoulos M, et al. Biochemical basis of hypoxic-ischemic encephalopathy. Neoreviews 2010; 11; e184-e193
4. Fanaroff and Martin’s Neonatal-Perinatal Medicine: Diseases of the Fetus and Infant, 9th edition. 2011, p
5. Marro, PJ, et al. Pharmacology review: Neuroprotective treatments for hypoxic-ischemic injury. Neoreviews 2010; 11; e311-e315
6. Zhang P, Moudgill N, Hager E, et al. Endothelial differentiation of adipose-derived stem cells from elderly patients with cardiovascular disease. Stem cells and development;20(6):
Objective
To determine whether hypothermia and hASC have additive neuroprotective effects in neonatal rats post HIE by comparing nissle staining of brain cortex thickness between treatments.