The Side Effects And Toxicity Of Hyperbaric Oxygen Tretment For Hypoxic-ischemic Brain Damage Neonatal Rats

Background: Hypoxic-ischemic encephalopathy (HIE) is anacquired disease due to asphyxia during early fetal or neonatal stages of anindividual leads to the damage of brain. In spite of major advances inmonitoring technology and knowledge of fetal and neonatal pathologies,perinatal asphyxia and HIE remains a serious condition, causing significantmortality and long-term morbidity. The incidence in most technologicallyadvanced countries of the world, such as USA is 2～4 cases per 1,000 births.However, in developing countries, the incidence of HIE is rather high. Butaccurate statistics is not available. In the China, it has been estimated tooccur in approximately 3～5 cases per 1,000 live-born at term and theincidence is higher in preterm neonates (1995). Between 10% and 60% ofneonates who experience a hypoxia-ischemia insult expire during thenewborn period and of those who survived resulting in behavioral andpsychological dysfunctions, such as motor or learning disabilities, cerebralpalsy, mental retardation and epilepsy or even death.Mechanisms of HIE remain unclear. The combined effects of cellularenergy failure, acidosis, excitatory amino acid and nitric oxide neurotoxity,Ca2+ accumulation, cellar phosphate lipid injury and free radical formationmay all serve to disrupt the structural of the nerve cells. As there is no effective treatment available for HIE currently, so scholars look forresolvent in succession in the near future. The promising therapeuticmethods for HIE include: (1) selective brain hypothermia, (2) hyperbaricoxygen. (HBO) treatment, (3) stem cell transplantation.HBO involves intermittent inhalation of 100% oxygen under apressure greater than 1 atmosphere absolute (ATA). At the present time, theanimal experiment and clinic investigation approve retinopathy ofprematurity (ROP) usually occurs after a prolonged exposure to normobarichyperoxia in newborn mammals and infants, especially in prematureneonates. At the same time, normobaric hyperoxia is a cause of thebronchopulmonary dysplasia (BPD) for neonates. Well then, whether theHBO for neonate HIE would induce the newborn infants occur ROP andBPD? Torbati (1995) employ 5-to-7-day-old Sprague-Dawley rats exposefor 5h either to 5 ATA oxygen. Two months after exposures, rats wereanesthetized, perfused intraventricularly with India ink, and retinal imageswere obtained. Retinal vascular density (RVD) in each image wascalculated as the number of pixels in the retinal vessel area divided by thetotal number of pixels in the image (retinal tissue and vessels). The RVDwas significantly increased from 0.0112±0.004 in the air-exposed controlsto 0.0417±0.029 in the HBO-exposed rats. He based these results educedthat a sustained HBO-induced retinal vasoconstriction in newborn ratsfollowed by a hypoxic-ischemic injury might result in vascularproliferation, thereby initiating ROP development on return to air. Calvert (2004) employ 7-day-old rat pups of HIBD treated with 100% oxygen at1ATA, 1.5 ATA, and 3.0 ATA for duration of 1h. He found there were nosigns of neovascularization after hyperoxia exposure and no abnormalitiesin the structure of the retina and no changes in the protein expression ofHIF-1a and vascular endothelial growth factor (VEGF) followinghyperoxia exposure. The results of above two investigators were conflictwith each other. Their HBO condition were not coincidence with that inclinical operate in China. The pressure of HBO for HIE is under 1.5～2ATA and the time is short of 1h (about 1h) presently. Under theseconditions whether the HBO for HIE would induce the newborn infantsoccur ROP and lung injury? At the same time whether the HBOT for HIEwould induce he newborn infants occur and present the other side effectsand toxicity?Herein, we sought out to determine whether HBO is able to offerneuroprotectivity against an HI insult and whether had side effects orcomplications and toxicity. PartⅠ: Effect of HBO on apoptosis of neural cells, retinal vascularproliferation and lung injury in neonatal hypoxia-ischemia neonatalrat modelObjective: To investigate the effect of apoptosis of neural cells,retinal vascular proliferation and lung injury after HBO treatment for HIBD.Methods: 7-day-old Sprague-Dauleys (SD) rats were randomly dividedinto six groups: (1) control group (CON, n=22); (2) HIBD group (HIBD,n=20), 7-day-old rat pups were subjected to unilateral carotid artery ligationfollowed by 2h of hypoxia (8% oxygen); (3) hyperbaric air group (HA,n=16); (4)normobaric hyperoxia group (HO, n=32); (5) hyperbaric oxygentreatment group (HBO, n=36); (6) normobaric hyperoxic+hyperbaricoxygen treatment group (2H, n=36). The HA and HBO group wereadministered with 2.0 ATA pressure of air and oxygen for duration of 1heach day after HIBD 2h, persist a series of 7 days. The HO group wasgiven 100% oxygen inhalation 7 days after HIBD. The 2H group was basedon HO group and HBO group. After a week, the rats were raised in normalpressure air for 7 days. All rats were killed in 21-day-old. The neural cellswere examined with terminal deoxynucleotidyltransferase mediated dUTPnick end labeling (TUNEL). The eyeballs and lungs were enucleated andfixed and then cross-sectioned, stained with hematoxylin and eosin (HE).The proliferated neovascular response was quantitated by counting thenuclei of endothelial cells of new vessels extending from the retina into thevitreous in sagittal cross sections. The rat retinae were flatted on the slides to assess the changes of retinal vessels after perfused intraventricularlywith ink. VEGF were determined on the cross-sections after immuno-histochemcal stain. Results: The number of TUNEL positive cells inhippocampal CA1 (mean±s) in same area of each visual field (total 12visual fields of each group), the CON group was 3±1, HIBD 156±21, theHA 135±14, HO 137±15, HBO 7±2 and 2H 26±4. contrasting with theCON group, the HIBD, HA, HO and 2H groups were markedly increased,P＜0.01. While there were not difference between the HBO, 2H gruop andthe CON group, P＞0.05. The nuclei of the new vessels of each group ineach slide (total 30 slides of each group) was: the CON group 12.5±3.2,HIBD 15.2±5.3, HA 13.7±4.2, HO 159.2±13.5, HBO 12.7±3.8, 2H208.3±16.6. The HO and 2H group were higher than the CON group, P＜0.001, but the difference were not significant between the CON and HIBD,HA and HBO group, P＞0.05. The changes of retinal vessels of flatteningretina on the slide show the HO and 2H groups increased significantly,especially in the 2H group. While there were no changes in evidence in theother groups. VEGF stain was found stronger in the inner retinal layer ofthe HO and 2H group than of the other groups. The changes of lung tissuestained with HE: the amount of alveoli pulmonis of HO and 2H groupsdecreased obviously, especially in the HO group. Intralobular interstitialthickening was markedly in the HO group, and was slightly in the 2Hgroups, no changes in the other groups. Conclusions: HBO may protect theHIBD brain tissue through decreasing neural cells apoptosis. It was hyperoxia rather than HBO (under 2.0 ATA) induced the retinal vesselproliferated and induced retinopathy of prematurity (ROP) occur andcaused the lung injury at the same time.PartⅡ: The effects of HBO on brain lipid peroxidation of neonatalrats after HBIDObjective: To investigate the effects of HBO on lipid peroxidation ofbrain tissue after HIBD. Methods: 7-day-old SD rats were randomlydivided into six groups: (1) Control group (CON, n=10); (2) HIBD group(HIBD, n=8); (3) hyperbaric air group (HA, n=8); (4) normobarichyperoxia group (HO, n=8); (5) hyperbaric oxygen treatment group (HBO,n=8); (6) normobaric hyperoxia+ hyperbaric oxygen treatment group (2H,n=8). The operations refer, to PartⅠ.The amount of8-iso-prostaglandinF2α(8-iso-PGF2α) in left brain homogenate (100mg)was detected by using the method of enzyme linked immunosorbent assay(ELISA). Results: The content of 8-iso-PGF2αof each group were: theCON group was 46.5±8.6 pg/g, HIBD 166.8±26.2pg/g, HA 185.5±36.5pg/g, HO 417.6±58.5pg/g, HBO 184.5±42.3 pg/g, 2H 352.5±43.4 pg/g. The8-iso-PGF2αcontent of the HIBD, HA, HO, HBO and 2H groups were allhigher than that of CON group, the difference were significant, P＜0.01.The 8-iso-PGF2αcontent of HO group was the highest in all groups. Therewere no difference between the HIBD group, the HA group and the HBOgroup, P＞0.05. In contrast with the HIBD group, the content of8-iso-PGF2αin the HO and 2H groups were increasing markedly, P＜0.01. Conclusions: The content of LPO of brain tissue is increased after HBIDand hyperoxia can aggravate the LPO degree. Hyperbaric air and HBOdoes not augment the effect of LPO farther in brain tissue after HIBD.PartⅢ: The clinical investigation of HBO treatment in neonates withHIEObjective: To investigate the effects and side-effcts of HBOtreatment in neonates with HIE. Methods: Forty-eight cases of HIEneonates were randomly divided into two groups: (1) Control group (CON,n=22), (2)HOB therapy group (HBO, n=26). Regular follow-up werecarried when these patients were at the age of 1-month-old, 3-month-old,6-month-old, 9-month-old, 1-year-old, 1.5-year-old, 2-year-old,2.5-year-old and 3-year-old. These children were given physicalexamination, optometry, eye fundus check, audiometry and so on. NBNAscore was given during the 1 month old and CDCC was given between 1month to 3 years old. When 1-month-old, 1-year-old and 3-year-old, thechild would be taken a plain chest radiography and head CT/MRI scan.Results: From June 2000 to June 2005, total 48 cases of HIE neonatesdistributing in eight hospitals were brought into the study. Before HBOT,the average NBNA score of CON group was 31.9±2.8 and the HBO groupwas 32.2±2.4, P＞0.05. After a series of HBO, the average NBNA scoreof HBOT group was 37.6±1.7 and was higher, than the CON groupsignificantly (33.2±2.1), P＜0.001. The results of CDCC of HBO group ineach age were: 6-M: 102.6±10.5, 1-Y: 106.5±12.2, 2-Y: 105.8±13.8, 3-Y: 113.6±13.4, all higher than the same ages of the CON group (6-M:84.9±11.6, 1-Y: 82.1±12.3, 2-Y: 85.2±11.4, 3-Y: 88.3±13.3) significantly, P＜0.001. Each group equality had a case died and a case of epileptic. In theHBO group, there was a case of glaucoma and a case of cataract and therewas no statistics significance between the two groups. There were 2 casesof cerebral palsy in the HBO group and 4 cases in the CON group, butthere was no statistics significance between the two group, P＞0.05. Therewere 3 cases of mental retardation in the HBO group and 8 cases in theCON group, there was difference between the two group, P＜0.05. Theresult of plain chest radiography and eye fundus check among the twogroup cases had no abnormities. Conclusions: HBO treatment in neonatalHIE can meliorate acute period symptom and improve long-term prognosis.HBO has no ROP and BPD side effects and the other complications. Underthe given pressure, HBOT for the neonatal HIE is a safe means.