Evaluating Neuroprotective Effects of Uridine, Erythropoietin, and Therapeutic Hypothermia in a Ferret Model of Inflammation-Sensitized Hypoxic-Ischemic Encephalopathy

Abstract

Perinatal hypoxic-ischemic (HI) brain injury, often in conjunction with an inflammatory insult, is the most common cause of death or disability in neonates. Therapeutic hypothermia (TH) is the standard of care for HI encephalopathy in term and near-term infants. However, TH may not always be available or efficacious, creating a need for novel or adjunctive neurotherapeutics. Using a near-term model of inflammation-sensitized HI brain injury in postnatal day (P) 17 ferrets, animals were randomized to either the control group (n = 43) or the HI-exposed groups: saline vehicle (Veh; n = 42), Ur (uridine monophosphate, n = 23), Epo (erythropoietin, n = 26), or TH (n = 24) to test their respective therapeutic effects. Motor development was assessed from P21 to P42 followed by analysis of cortical anatomy, ex vivo MRI, and neuropathology. HI animals took longer to complete the motor assessments compared to controls, which was exacerbated in the Ur group. Injury resulted in thinned white matter tracts and narrowed cortical sulci and gyri, which was mitigated in Epo-treated animals in addition to normalization of cortical neuropathology scores to control levels. TH and Epo treatment also resulted in region-specific improvements in diffusion parameters on ex vivo MRI; however, TH was not robustly neuroprotective in any behavioral or neuropathological outcome measures. Overall, Ur and TH did not provide meaningful neuroprotection after inflammation-sensitized HI brain injury in the ferret, and Ur appeared to worsen outcomes. By comparison, Epo appears to provide significant, though not complete, neuroprotection in this model.

Keywords: asphyxia; erythropoietin; neonatal; therapeutic hypothermia (TH); uridine.

Figure 1

Body weight analysis. (A) At P42, all Ur animals were significantly lighter than control animals. In the males, the other HIH animals were also lighter than the controls; however, the comparison between control males and Veh males was not significant. In the females, the Veh animals were significantly lighter than the control animals, but TH and Epo animals were more comparable to the control animals. (B) On the day of HIH (P17), mean (SD) weight of the male kits was 93.0 (7.8) g. On average, HIH males lost 9.6% (7.1%) of their body weight between P17 and P18 as a result of the HIH insult. By P19, HIH males gained 2.0% (6.9%) of their P17 body weight back, compared to a 26.7% (6.0%) weight gain in controls by P19. The HIH males never fully recovered from the weight loss that followed HIH, but the difference in weight between Veh males and control males on P42 lost its significance. (C) At P17, the mean weight of the female kits was 83.3 (5.6) g. On average, HIH females lost 11.3% (8.3%) of their body weight between P17 and P18 as a result of the HIH insult. By P20, HIH females gained 4.4% (13.5%) of their P17 body weight back, compared to a 25.2% (9.0%) weight gain in controls by P20. The HIH females took an extra day longer to gain back the P17 body weight deficit when compared to the HIH males, but the HIH females maintained a weight gain pattern more consistent with the control females by P42. * Denotes p–value < 0.05, ** denotes p–value < 0.01, and ns denotes no significant difference.

Figure 2

Motor development. (A) AUC analysis of the total time (median; IQR) to complete all reflex tests showed that Ur animals (8263.3 s · days; 7782.8–8658.0 s · days) took significantly longer to complete reflex testing compared to control animals (8083.3 s · days; 7690.5–8486.2 s · days) throughout the testing period. (B) Frequency of open field corner visits in the Veh, Ur, Epo, and TH groups relative to the control group. Median corner visit frequency in controls was 49. Median values were significantly increased in the Ur group by 45.5% and non–significantly increased in all other HIH–exposed groups relative to control median. (C) Time in seconds (s) spent not moving in open field in the Veh, Ur, Epo, and TH groups relative to the control group. Median time spent not moving was 99.7 s in the control group and significantly increased in the Ur group by 50.7%. In all other HIH groups, median time spent not moving was non–significantly increased. (D) Hindlimb BOS in the Veh, Ur, Epo, and TH relative to the control median. Median hindlimb BOS was 0.01 cm/g in the control group and was significantly increased in the Ur, Epo, and TH groups by 20.3, 12.5, and 18.0%. (E) Foreprint area in the Veh, Ur, Epo, and TH groups relative to the control median. Median foreprint area was 0.39 cm/g in the control group and significantly increased by 35.6% in the Ur group. In all other HIH–exposed groups, median foreprint area was non–significantly increased. (F) Number of attempted runs in the control, Veh, Ur, Epo, and TH groups. The median number of attempted runs was 6 in the control group and significantly increased to 8 in the Ur group. All other HIH groups had non–significant increases in number of attempted runs. * Denotes p–value < 0.05, ** denotes p–value < 0.01, and ns denotes no significant difference.

Figure 3

Cortical Pathology and Morphology. The median and IQR is plotted on each graph. (A) Surviving animals received combined scores of 0–8 (0–4 cortical pathology + 0–4 mineralization) while animals that died after the HIH insult received a score of 8.5. Control animals had a median cortical pathology score of 0 with no deaths. Median scores significantly increased in the Veh group to 1 and in the Ur and TH groups to 1.5. Deaths also increased in the Veh, Ur, and TH groups to 4, 5, and 3, respectively. Compared to controls, number of deaths in the Epo group increased to 2. (B) Summed sulci length in the HIH groups relative to the control group. After calculating the sum of the lengths of each sulci, the HIH groups all displayed a shorter sum of sulcal length than the control group. On average, HIH brains sulci were 8.8% (3.3–16.9%) shorter than control brains. (C) Summed gyri widths in the HIH–exposed groups relative to control median. Similar to the summing of sulci, the summing of the width of each gyri showed that control brains had significantly wider gyri than Veh (3.6%; 0.1–9.5%), Ur (7.3%; 1.4–15.6%) and TH (5.4%; 2.6–8.4%) brains. Epo brains exhibited summed gyral widths more similar to control brains. * Denotes p–value < 0.05, ** denotes p–value < 0.01, *** denotes p–value < 0.001, **** denotes p–value < 0.0001, and ns denotes no significant difference.

Figure 4

Diffusion Tensor Imaging. Comparison of FA values throughout the white matter (blue). Light red indicates p < 0.15 and dark red indicates p < 0.05 after threshold-free cluster enhancement (TFCE) adjustment for multiple comparisons. (A) Contol FA values greater than Veh FA values in the dorsal striatum and substantia innominata. (B) TH FA values greater than Veh FA values throughout cerebral white matter. (C) Epo FA values greater than Veh FA values in the anterior cerebral white matter and interior capsule. (D) Control FA values greater than Epo FA values in the peri-hippocampal cerebral white matter tracts.

Figure 5

Quantitative IHC. (A) After measuring the subcortical white matter (SWM) tract thickness of the area inferomedial to the lateral sulcus in the GFAP–stained slices, the length of the WM tracts (median % difference; IQR) was significantly decreased in Veh (15.4%; 5.1–31.8%), Ur (20.2%; 6.3–31.3%), and TH (25.6%; 12.5–42.8%) brains when compared to controls. (B) In the three regions of interest (ROIs) in the white matter measured, the summed measurements of tract thickness were significantly thinner in Veh (15.6%; 1.6–30.0%), Ur (32.0%; 26.1–48.3%), and TH (27.6%; 15.2–33.6%) brains when compared to control brains. Epo brains had white matter thickness more consistent with controls in all ROIs. (C) Compared to controls, the corpus callosums (CC) of the Veh (21.4%; 3.9–40.6%), Ur (32.6%; 17.0–47.3%), Epo (25.1%; 12.7–44.9%), and TH (41.0%; 13.6–52.3%) brains were significantly thinner when compared to the CC of control brains. * Denotes p–value < 0.05, *** denotes p–value < 0.001, **** denotes p–value < 0.0001, and ns denotes no significant difference.

Figure 6

P17 Model Timeline. On P17, animals were administered 3 mg/kg LPS before undergoing bilateral carotid artery ligation, with the RCA occlusion being reversed after the gas exposure (hypoxia-hyperoxia-hypoxia). Special care was taken to isolate the artery away from surrounding vessels and nerves. Treatment was then initiated within a 90–120 min time window after the end of the HIH insult. Basic reflexes were assessed from P21 to P32 followed by a battery of late behavioral testing on P42. The brains were then extracted and processed for further evaluation.