Concurrent erythropoietin and hypothermia treatment improve outcomes in a term nonhuman primate model of perinatal asphyxia

Abstract

Background: Up to 65% of untreated infants suffering from moderate to severe hypoxic-ischemic encephalopathy (HIE) are at risk of death or major disability. Therapeutic hypothermia (HT) reduces this risk to approximately 50% (number needed to treat: 7-9). Erythropoietin (Epo) is a neuroprotective treatment that is promising as an adjunctive therapy to decrease HIE-induced injury because Epo decreases apoptosis, inflammation, and oxidative injury and promotes glial cell survival and angiogenesis. We hypothesized that HT and concurrent Epo will be safe and effective, improve survival, and reduce moderate-severe cerebral palsy (CP) in a term nonhuman primate model of perinatal asphyxia.

Methodology: Thirty-five Macaca nemestrina were delivered after 15-18 min of umbilical cord occlusion (UCO) and randomized to saline (n = 14), HT only (n = 9), or HT+Epo (n = 12). There were 12 unasphyxiated controls. Epo (3,500 U/kg × 1 dose followed by 3 doses of 2,500 U/kg, or Epo 1,000 U/kg/day × 4 doses) was given on days 1, 2, 3, and 7. Timed blood samples were collected to measure plasma Epo concentrations. Animals underwent MRI/MRS and diffusion tensor imaging (DTI) at <72 h of age and again at 9 months. A battery of weekly developmental assessments was performed.

Results: UCO resulted in death or moderate-severe CP in 43% of saline-, 44% of HT-, and 0% of HT+Epo-treated animals. Compared to non-UCO control animals, UCO animals exhibit poor weight gain, behavioral impairment, poor cerebellar growth, and abnormal brain DTI. Compared to UCO saline, UCO HT+Epo improved motor and cognitive responses, cerebellar growth, and DTI measures and produced a death/disability relative risk reduction of 0.911 (95% CI -0.429 to 0.994), an absolute risk reduction of 0.395 (95% CI 0.072-0.635), and a number needed to treat of 2 (95% CI 2-14). The effects of HT+Epo on DTI included an improved mode of anisotropy, fractional anisotropy, relative anisotropy, and volume ratio as compared to UCO saline-treated infants. No adverse drug reactions were noted in animals receiving Epo, and there were no hematology, liver, or kidney laboratory effects.

Conclusions/significance: HT+Epo treatment improved outcomes in nonhuman primates exposed to UCO. Adjunctive use of Epo combined with HT may improve the outcomes of term human infants with HIE, and clinical trials are warranted.

© 2013 S. Karger AG, Basel.

Figures

Figure 1

Schedule of procedures and assessments for the nonhuman primate model of perinatal asphyxia. Abbreviations: UCO, umbilical cord occlusion; MRI, magnetic resonance and diffustion tensor imaging and spectroscopy; PT, physical therapy; EEG, amplitude-integrated electroencephalography. * Note, the first MRI scan was performed at either 24 or 72 hours of age.

Figure 2

Death or severity of cerebral palsy (CP) after umbilical cord occlusion (UCO). Top panel shows the number of animals with death or moderate/severe CP, and the bottom panel shows the individual severity scores for all animals by treatment group. Severity of CP was based upon four physical therapy exams performed at one week, and at 1, 4, and 8 months of age. UCO increased the rate of death or moderate/severe CP as compared to controls (ANOVA * = p

Figure 3

Initial response to umbilical cord occlusion (UCO). Data are mean ± SEM Apgar scores for neonatal animals separated by the duration of UCO. Controls are gray boxes, 15 min UCO as black boxes (dashed line), and 18 min UCO as black diamond (solid line).

Figure 4

Longitudinal growth of nonhuman primates. The main graph plots the average weight of UCO-exposed animals (lines) superimposed upon the individual daily weights of control animals (gray squares) with an inset graph plotting the mean (± SEM) days to regain birthweight. Mean daily weights for UCO saline (black line), UCO HT (blue line) and UCO HT+Epo (red line) groups are shown separately. The inset shows that the time to regain birthweight was significantly elevated for UCO saline and UCO HT animals (Dunnett’s test, † = p<.01 these graphs illustrates that uco reduces the growth of animals despite caloric adjustments to foster weight gain.>

Figure 5

Cerebellar growth from birth to 9 months of age. Box and whisker plots (median and quartile range) illustrate the percentage increase in cerebellar volume for animals in each treatment group. Cerebellar growth was decreased by umbilical cord occlusion (UCO) compared to controls. Significant differences are indicated (ANOVA * = p≤.05). Treatment with hypothermia (HT) plus erythropoietin (Epo) is associated with improved growth after UCO when compared to UCO saline treated animals (Dunnett’s t, bar p=0.019, N=9 UCO saline, UCO 6 HT, and 8 UCO HT+Epo).

Figure 6

Diffusion tensor imaging after umbilical cord occlusion (UCO). TBSS analysis of mode of anisotropy, relative anisotropy and raw T2 signal maps with red indicating areas that had increased signal compared to UCO saline and blue areas indicating lower signals. Highlighted (red or blue) regions p ≤ 0.05 using Threshold-Free Cluster Enhancement with correction for multiple comparisons. Higher values of mode of anisotropy and relative anisotropy are associated with healthy white matter. Lower values of T2 signal are associated with healthy white matter. Top two panels show validation of the perinatal asphyxia model and the bottom panel demonstrating that therapeutic hypothermia (HT) plus erythropoietin (Epo) improves diffusion tensor imaging.

Figure 7

Diffusion tensor imaging (DTI) after 18 min of umbilical cord occlusion (UCO) .TBSS analysis of fractional anisotropy, relative anisotropy and volume ratio between UCO saline and hypothermia plus erythropoietin (HT+Epo) after 18 min of UCO is shown. DTI scans occurred within the first 72h. Red areas indicate where HT+Epo group had higher values than UCO saline while blue signifies lower values in the HT+Epo group. Highlighted regions (red or blue) p ≤ 0.05 using Threshold-Free Cluster Enhancement with correction for multiple comparisons. Higher values of fractional anisotropy and relative anisotropy are indicative of healthy white matter. Lower values of volume ratio are indicative of healthy white matter.

Figure 8

Pharmacokinetics of erythropoietin (Epo) in the setting of hypothermia. Panel a shows the peak erythropoietin concentrations (Cmax) of the ten animals treated with hypothermia and Epo (HT+Epo) divided into outcomes of no cerebral palsy (CP) or mild CP. Panel b illustrates the paramater area under the curve (AUC). Panel c shows mean (± SEM) plasma concentrations of Epo in the first 72 hours. Epo concentrations of non-Epo-treated asphyxiated animals are shown by white diamonds. Pharmacokinetics of high dose Epo are depicted as black circles while the lower dose Epo is shown as gray squares. Epo dosing occurred at times 0, 24 and 48 hours.