| Neonatal hypoxic and ischemic encephalopathy (HIE) is a disease with nervous system damage that is caused by hypoxic and ischemic (HI) factors during perinatal period. HIE, one of the major causes leading to newborn nerve system damage, is characterized by its high mortality and sequelae such as amentia, brain palsy and epilepsy. HIE study has become one of the major focus areas of perinatal medicine research.NGF is implicated in the survival, immigration, growth and differentiation of nerve cells, and the functional establishment of nerve cells with other types of cells. Under physiological condition, little NGF can be detected in the brain. On the other hand, many studies have reported the elevated expression of NGF resulting from different brain damages. Emergency expression of endogenous NGF may play a protective role, however, this elevated expression is too short-lasting to produce any effective protection. The regenerative action of exogenous NGF is well known, and one Chinese report has shown that exogenous NGF administered by intraventricular injection can reduce apoptosis in the brain of neonatal rats with hypoxic-ischemic brain damage. However, it is controversial whether NGF can pass blood brain barrier (BBB) and therefore NGF can be used to treat HIE. There are several approaches for using exogenous NGF in the treatment of central nervous system diseases, including NGF infusion into brain, explantation of NGF-producing cells into damaged neurocytes, and gene therapy. These methods, however, have not been widely used clinically due to various disadvantages. Therefore, further study of the expression and protective mechanism of NGF is needed. In this study, we investigated the permeability and distribution of peripherally administered NGF and its protection on hypoxic-ischemic brain damage. Our findings have provided a new potential means for the clinical therapy of HIE.There are four parts in this study. Part I: by using peripherally administered 125I-labeled NGF, we examined the blood brain barrier permeability and peak time of NGF in different ages of neonatal rats with hypoxic-ischemic brain damage (HIBD), and its relationship with 125I-NGF level in the blood. Part II: we used radioiodination and autoradiography, and measured the 125I-NGF level in basal forebrain, cortex, hippocampus, thalamus, cerebellum of the neonates with HIBD, and determined the absorption and distribution in different brain issues. Part III: we observed the change of moisture capacity, apoptosis and synaptophysin in rat brain tissues by using immunohistochemistry and TUNEL staining, and demonstrated the protective effect of NGF in neonatal rats with hypoxic brain damage. Part IV: we evaluated a marker indicating the severity of HIE and its detection and treatment, and tested the clinical effect of NGF in HIE and determined the best time point of therapy in some patients with moderate and serious HIE.The results show:1. 125I-βNGF administered peripherally was detected in all the rat brains tested, indicating that NGF can pass blood brain barrier (BBB) and reach brain tissues. The peak time of NGF penetration is 30 minutes.2. Both the brain 125I-βNGF level in neonatal rats and its ratio to the blood 125I-βNGF level (RBB) is higher than in adult rats, and neonatal rats with HIBD possess even higher brain 125I-βNGF level and RBB than normal neonates. These observations showed that BBB transmissivity of neonates is higher than that of adult rats, probably due to the growth flaw of BBB, and hypoxia might increase its permeability.3. Absorptions of 125I-NGF in the basal forebrain, cortex, hippocampus and cerebellum of both normal neonatal rats and neonates with HIBD were significantly higher than that of 125I in these brain regions, indicating that NGF can pass BBB and reach these brain regions. Absorptions in the cortex, hippocampus and cerebellum of hypoxic group (125I-NGF) were significantly higher than that of control group (125I-NGF) suggesting that permeability of NGF of the blood brain barriers of newborn rats with hypoxidosis was increased, further supporting that NGF can penetrate the BBB of neonatal rats with HIBD.4. Brain tissue moisture capacity of neonatal rats 72 hours after HIBD was higher than that of normal neonatal rats. No significant difference between NGF-treated group and normal control group was detected. However, brain tissue moisture capacity of NGF-treated neonates was lower than HIBD neonates. These findings suggested that NGF treatment could alleviate brain edema in HBID rats.5. Body weight of NGF-treated HIBD neonates increased faster than those without NGF treatment suggesting that NGF could relieve the clinical HIBD symptoms and help recovery.6. Synaptophysin level in the frontal cortex and CA1 subregion of hippocampus of neonatal rats with HIBD decreased significantly following HI, whereas NGF treatment significantly increased synaptophysin level in neonates with HIBD, implicating NGF in CNS regeneration and reparation.7. TUNEL staining positive cells in the brains of NGF-treated neonates is lower than those without NGF treatment, suggesting that NGF could decrease apoptosis and alleviate brain damage.8. 48 and 72 hours following moderate HIE, the concentration of cerebrospinal fluid neuron specific enolase(CSF-NSE) was significantly increased, and it recovered to the basal level in 7 days. We found, however, that serious HIE led to a slower recovery of CSF-NSE, with abnormal CSF-NSE concentration detected in some cases even after 7 days following serious HIE. Therefore, CSF-NSE level can be used as an early marker for the severity and effect of HIE treatment. Concentration of CSF-NSE at the time points of 48 and 72 hours can be used for the early HIE damage diagnosis, and CSF-NSE concentration at the time point of 7 days for the measurement of brain damage recovery.9. The concentration of CSF-NSE was lower in the HIE rats with 7 days of NGF treatment than that in those without NGF treatment. Neonatal Behavioral Neurological Assessment (NBNA) at 14 and 28 days of postnatal age, and Children's Developmental Center of China (CDCC) test at 4 months of postnatal age were significantly increased in NGF-treated groups compared with control groups (those without NGF treatment). These findings suggest that NGF can reduce the brain damage during acute and recovery period in HIE.10. CDCC score in 4 months after 3 days of NGF treatment was remarkably higher than that in 14 days subjected to same treatment, suggesting that earlier NGF treatment is very critical in HIE.To summarize, peripherally administered NGF can penetrate the blood brain barrier and reach brain tissues, reduce brain edema, cell apoptosis or necrosis, and promote synapse regeneration in neonatal rats with hypoxiaischemia brain damage, which might underlie the protective effect of NGF in brain damage. Our study has added new insights into the mechanism of HIE, and provided new approach for the effective treatment of HIE patients.The original findings and novelty of present study: so far most studies on BBB mainly focus on adult rats, and little research is performed by using neonatal rats. This research for the first time in China has studied the permeability, distribution and protective effect of nerve growth factor administered peripherally in neonatal rats with HIBD, and performed the clinical test. Our findings will open novel means for the effective therapy of HIE patients.
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