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. Author manuscript; available in PMC: 2024 Feb 1.
Published in final edited form as: Neurochem Int. 2022 Dec 30;163:105471. doi: 10.1016/j.neuint.2022.105471

Selectively Compromised Inner Retina Function following Hypoxic-Ischemic Encephalopathy in mice: a noninvasive measure of severity of the injury

Onur E Taparli 1,2,*, Pawan K Shahi 1,3,*, Nur Sena Cagatay 1,2, Nur Aycan 1, Burak Ozaydin 4, Sefer Yapici 1,2, Xinying Liu 1, Ulas Cikla 1,4, Dila Zafer 1,2, Jens C Eickhoff 5, Peter Ferrazzano 1,2, Bikash R Pattnaik 1,3,6,*, Pelin Cengiz 1,2,*
PMCID: PMC9905320  NIHMSID: NIHMS1866850  PMID: 36592700

Abstract

The intricate system of connections between the eye and the brain implies that there are common pathways for the eye and brain that get activated following injury. Hypoxia-ischemia (HI) related encephalopathy is a consequence of brain injury caused by oxygen and blood flow deprivation that may result in visual disturbances and neurodevelopmental disorders in surviving neonates. We have previously shown that the tyrosine receptor kinase B (TrkB) agonist/modulator improves neuronal survival and long-term neuroprotection in a sexually differential way. In this study, we tested the hypotheses that; 1) TrkB agonist therapy improves the visual function in a sexually differential way; 2) Visual function detected by electroretinogram (ERG) correlates with severity of brain injury detected by magnetic resonance (MRI) imaging following neonatal HI in mice. To test our hypotheses, we used C57/BL6 mice at postnatal day (P) 9 and subjected them to either Vannucci's rodent model of neonatal HI or sham surgery. ERG was performed at P 30, 60, and 90. MRI was performed following the completion of the ERG. ERG in these mice showed that the a-wave is normal, but the b-wave amplitude is severely abnormal, reducing the b/a wave amplitude ratio. Inner retina function was found to be perturbed as we detected severely attenuated oscillatory potential after HI. No sex differences were detected in the injury and severity pattern to the retina as well as in response to 7,8-DHF therapy. Strong correlations were detected between the percent change in b/a ratio and percent hemispheric/hippocampal tissue loss obtained by MRI, suggesting that ERG is a valuable noninvasive tool that can predict the long-term severity of brain injury.

Keywords: electroretinogram (ERG); hypoxia-ischemia (HI); brain-derived neurotrophic factor (BDNF); tyrosine receptor kinase B (TrkB); 7,8-dihydroxyflavone (7,8-DHF); electroretinogram (ERG)

1. INTRODUCTION

Neonatal hypoxia-ischemia (HI) related brain injury leads to severe, life-long morbidities and mortalities in thousands of neonates and children each year. The estimated incidence of neonatal HI is 2.5 per 1000 live births (Volpe 2008). Nearly half of the term infants suffering severe HI die within weeks after birth, and 25% of those surviving exhibits permanent neuropsychological sequelae (Graham, Ruis et al. 2008). (Ferriero, 2004, Nelson and Lynch, 2004, Drobyshevsky, et al., 2007, Hill and Fitch, 2012).

Neonatal HI has been extensively investigated for its effects on motor and cognition, but little has been done to examine its impact on visual abnormalities. Although cortical visual dysfunction is a significant cause of visual impairment, the immature retina is also highly susceptible to HI injury (Huang, Huang et al. 2012, Zaitoun, Cikla et al. 2018). The developing retinal tissue is highly metabolic and requires abundant oxygen and glucose. Due to its high-energy demand, the developing retina is especially vulnerable to damage caused by oxidative stress in the aftermath of HI. Clinical studies showed that the visual function of infants post-HI correlated with their neuromotor and global development (Mercuri, Haataja et al. 1999). Neonatal HI can also cause cortical or cerebral visual impairments that affect children's learning and social interaction (Chokron, Kovarski et al. 2021). While therapeutic hypothermia is the only accepted standard therapy after HI in human neonates, profound alterations in the retinal vasculature indicate the importance of developing therapeutic strategies to alleviate the retinal injury post-HI.

Retinal ganglion cells (RGCs) are inner retinal neurons that receive visual signal input from photoreceptors and bipolar cells and transmit it to the visual cortex. The studies show that brain-derived neurotrophic factor (BDNF) activity, through binding to its receptor, tyrosine receptor kinase B (TrkB), plays a vital role in the development and function of the neurons (Park and Poo 2013). Changes in levels of BDNF are associated with retinal disorders because most RGCs contain BDNF and its high-affinity TrkB receptor. Gryciuk et al. showed that increased BDNF signaling led to long-term retinal ganglion-cells neuroprotection in the retina's central and peripheral regions after glaucoma (Wojcik-Gryciuk, Gajewska-Wozniak et al. 2020). However, the limited systemic delivery of BDNF makes it challenging to use in the clinical setting. Alternatively, the small molecule 7,8-dihydroxyflavone (DHF) has a selective affinity for TrkB and can cross the blood-brain barrier (Jang, Liu et al. 2010). Recent animal investigations on the neuroprotective benefits of 7,8-DHF therapy in the visual system reveal that 7,8-DHF preserves retinal functionality and protects RGCs in adult retinas with optic nerve transection (Gallego-Ortega, Vidal-Villegas et al. 2021) and in immature retinas with HI injury (Huang, Huang et al. 2021).

We have recently shown that 7,8 DHF treatment has neuroprotective effects following HI in the hippocampus of female mice (Cikla, et al. 2016). In this study, we aim to investigate the long-term effects of neonatal HI and 7,8-DHF therapy on the retinas of adolescent and young adult male and female mice using ERG and correlate the ERG findings with the severity of brain injury obtained by magnetic resonance imaging (MRI).

2. MATERIALS AND METHODS

2.1. Ethical statement:

All procedures involving the use of animals in ophthalmic and visual research conformed to the ARVO declaration. All animals used in experiments were cared for in accordance with a protocol authorized by the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin School of Medicine and Public Health, which is certified by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All animals are maintained in tightly controlled temperature (23 ± 5°C), humidity (40-50%), and light/dark (12/12 h) cycle conditions in a 200 lux light environment.

2.2. Induction of neonatal HI:

We used a modified Vannucci model to induce HI (Vannucci and Vannucci, 1997). C57BL/6J mice were anesthetized with isofluorane (Butler Schein Animal Health Supply; 5% for induction, 2% for maintenance) in 3:1 nitrous oxide-oxygen on a postnatal day (P) 9. Animals were kept on a surgical table heated to 36°C so that their body temperatures would remain constant for the duration of the procedure. Using a surgical microscope (Nikon SMZ-800 Zoom Stereo, Nikon), a midline skin incision was made, and the muscle overlying the trachea was exposed. An electrical cauterization was followed by a blunt dissection to free the left common carotid artery from the carotid sheath. We then injected 0.5% bupivacaine into the incision and closed it with a single 6.0 silk suture. Animals were returned to their colony to recover for 2 hours in the normoxic chamber at 36.5°C and continuously monitored. The animals were then placed in a hypoxic chamber (BioSpherix) equilibrated with 10% O2 and 90% N2 at 36°C for 50 min to cause unilateral (left-sided) hypoxic-ischemic-reperfusion injury. After hypoxic exposure, mice were placed again in 36.5°C normoxic chamber for 10 minutes recovery period. Sham-operated mice received the same amount of anesthesia for the same duration and exposure of the left common carotid artery without electrocauterization or hypoxia as previously described (Cikla, et al. 2016).

2.3. Drug administration:

Male and female littermates were randomly assigned to one of three groups: sham-vehicle control, HI-vehicle control, or the HI + 7,8-DHF therapy groups. 7,8-DHF was dissolved in DMSO at a concentration of 3 mg/mL and put into aliquots that could be frozen for up to a month. On the day of administration, the DHF was diluted to 0.1 mg/mL with sterile PBS and injected intraperitoneally at a final concentration of 5 mg/kg. The HI + 7,8-DHF-treated mice received the initial dose of the drug 10 min after induction of HI. Following that, mice received the same daily dose of 7,8-DHF (5 mg/kg) for 6 days, for a total of 7 doses. The HI-vehicle control groups and sham mice received an equal volume of PBS simultaneously for the same duration. 7,8-DHF were obtained from Sigma (St. Louis, MO).

2.4. Electroretinography (ERG)

After going through the sham and HI procedures at P9, as described in Fig. 1, three weeks old mice were transferred to the facility where ERG is performed. Mice were placed in a room with controlled temperature, humidity, and a light-dark cycle to help them adjust to their environment. ERG was performed on male and female mice at P30, P60, and P90. ERG was carried out as described previously following dark adaptation (Zaitoun and Sheibani 2021). In brief, under low red lighting, intraperitoneal injections of ketamine (80 mg/kg) and xylazine (16 mg/kg) were used to anesthetize mice. A drop of 0.5% proparacaine hydrochloride was given topically for local anesthetic, and a 1% tropicamide eye drop was used to dilate the pupil. Animals were kept on a 37°C heating pad under anesthesia to prevent hypothermia. A 2% hypromellose (GONIOVISC, HUB Pharmaceuticals, LLC, Rancho Cucamonga, CA) eye drop was placed on the cornea to keep it moistened to provide electrical contact with a 2.5 mm contact lens ERG electrode. Espion system (Diagnosys LLC, MA) was used to record ERG from mouse eyes, and Espion color dome Ganzfeld was employed to illuminate the eyes uniformly. The reference electrode was inserted through the cheek, whereas the ground electrode was inserted subcutaneously on the back near the tail. The eyes were exposed to a sequential increment of flash intensities (0.1 to 30 cd.s/m2) for 400 ms with a 2 s interval between each flash. The data thus collected were analyzed using the Espion software (Diagnosys LLC, MA) and Origin2018b (Origin Lab Corp., MA. Each eye's a-wave was measured from the baseline to the negative peak, and b-wave amplitudes were calculated from the peak of the a-wave to the peak of the positive waveform. The b/a ratio was determined by dividing the b-wave amplitude by the a-wave amplitude. The same mouse's HI-injured eye (left eye) is compared and normalized with the control eye (right eye). Percent change in the b/a ratio is calculated and compared between the sham, HI, and HI+ treated males and females. In addition, the percent change in the b/a ratio correlates with the percent hippocampal and hemispheric loss obtained by T2 MRI.

Figure 1:

Figure 1:

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Experimental Design: Mice were exposed to HI at postnatal day 9 (P9). Mice were treated with 7,8-DHF or VC starting 10 min from the HI for 7 days. Electroretinogram (ERG) was used to assess the retinal function of mice at days 30, 60, and 90. Following ERG, T2-weighted MRI was performed at P90+ to determine the extent of brain injury.

2.5. MRI Parameters and Post-Processing

In order to assess the extent of brain injury, T2-weighted MRI was performed following ERG testing after P90. MRI was performed using an Agilent 4.7-tesla Small Animal MRI scanner with a Varian 200-MHz quadrature mouse radiofrequency coil (38 mm internal diameter) subsequently to ERG records at P90. Mice were anesthetized with 1.5% isoflurane in an oxygen/air mixture administered through a nose cone. Then the skull was immobilized in a cradle position within the center of the magnet bore. The respiratory rate and body temperature were monitored with a monitoring unit, and the temperature was maintained within physiologic limits (37.0 ± 0.2°C) with a stream of warm air in the magnet bore. T2-weighted fast spin echo images (TR = 3,500 ms; effective TE = 60 ms; echo train length = 8; matrix size: 192 × 192; averages = 8) were acquired from 14-16 contiguous axial slices with a field of view of 20 × 20 mm and a slice thickness of 0.8 mm. T2-weighted MRI scans were manually assessed for artifacts. Rician noise was removed from T2-weighted scans using an adaptive non-local means algorithm as part of the Advanced Normalization Tools software suite (Manjón, Coupé et al. 2010, Tustison, Cook et al. 2021). Scans were corrected for inhomogeneous spatial intensities using the N4BiasFieldCorrection tool (Tustison, Avants et al. 2010).

2.6. T2-weighted MRI to quantify percent tissue injury

The T2-weighted MRI percent tissue loss quantification was manually carried out by using ITK-SNAP. On average, 9 slices were used for hemispheric measurements out of 14 slices. To analyze T2-weighted MRIs for percent volume loss, images were manually segmented using ITK-SNAP. Specifically, preprocessed T2-weighted MRIs were loaded into the ITK-SNAP viewer. Only viable tissue was labeled for volumetric analysis, with lesions, cysts, or tissues with liquefaction excluded. Manual segmentations of the brain's entire right and left hemispheres were additionally labeled to account for cortical damage. After segmentation, volumes of the manually labeled brain hemispheres were automatically calculated in ITK-SNAP by multiplying the voxel count in each slide by the voxel volume (0.004883 mm3). The volume loss was converted into a percent change using the following formula: [(CL Volume) - (IL Volume) ÷ (CL Volume)] × 100 (Ozaydin, Bicki et al. 2022).

2.7. Statistical analysis

The distributions of percent changes in b/a ratios in males and females at each time point were non-normally distributed after examining normal probability plots and histograms. Therefore, a nonparametric Wilcoxon rank sum test was used to compare the % changes in the b/a ratios in males and females between experimental conditions, i.e., Sham vs. HI, Sham vs. HI+DHF and HI vs. HI+DHF. Analogously, a nonparametric Wilcoxon rank sum test was used to compare the % change in b/a ratios between groups for males and females combined. Linear regression analyses were conducted to evaluate the correlations between ERG and MRI measurements in males and females at each time point. Temporal changes in the % change in the b/a ratios were analyzed using a nonparametric Wilcoxon signed-rank test. All reported p-values are two-sided, and P<0.05 was used to define statistical significance. Statistical analyses were conducted using SAS software (SAS Institute, Cary, NC), version 9.4. All data are reported as the mean (average value) and the variability (SEM). We used two-sample t-tests for comparison, and a P value <0.05 was interpreted as significant.

3. RESULTS

3.1. Electroretinogram (ERG) as a method of detecting retinal injury in a mouse model of neonatal HI

The physiological light response can be measured as an ERG widely used in clinical practice as an objective diagnostic test (Robson, Frishman et al. 2022). Photoreceptors in the retina are hyperpolarized in response to phototransduction (light activation) which is represented as the first negative waveform of the ERG as a-wave recorded at the eye's surface (Pinto, Invergo et al. 2007) (Kinoshita and Peachey 2018). The a-wave response is followed by depolarization of the bipolar neurons, referred to as the b-wave response. As stated previously, the HI condition and occlusion of the left common carotid artery in the mouse model used in this study resulted in injury to the eye ipsilateral to the carotid artery occlusion (the left eye). Our results are comparable with other rodent studies that found the inner retina, which generates a b-wave, is significantly more vulnerable than the outer retina, which produces a wave (Zaitoun and Sheibani 2021). However, as in humans, despite the same age, sex and insult, the HI results in variable retinal injury and ERG responses.

As shown in Figures 2 A, B, and C, the average response of the left eye a-wave of female mice with mild HI was 252.6 ± 31.16 μV, while the average response of the control (right) eye was 225.56 ± 39.26 μV (p=0.6). The amplitude of the female b-wave in our mild category is 411.2 ± 51.04 μV, which is not much less than the amplitude of the control eye, which is 456.72 ± 73.03 μV (p=0.62). The male mice also showed similar responses for both a-wave and b-wave, with measurements of left eye a-wave 226.05 ± 23.66 μV compared to 201.16 ± 16.21 μV for the control (right) eye (p=0.4) and 429.35 ± 37.04 μV for the left eye b-wave compared to 467 ± 43.12 μV for the control eye (p=0.52) (Fig 3 A, B, and C). Both female and male mice categorized as moderate showed reduced b-wave amplitude in the injured left eye compared to the control eye. For the injured female eyes, b-wave measured 384.35 ± 74.74 μV compared to 441.75 ± 62.37 μV (p=0.52) for the control eye. The b-wave of the injured male eye also had a similar decrease that measured 388 ± 33.22 μV compared to 478.27 ± 43.71 μV (p=0.11) for the control eye [Fig 2 and 3 (A-C)]. There was a slightly increased a-wave amplitude in the injured eyes for females and males compared to the control eyes. The female injured a-wave amplitude measured 224.1 ± 41 μV, and the male a-wave measured 234.75 ± 19.51 μV compared to the control eyes 191.82 ± 24.63 μV (p=0.52) and 223.57 ± 14.65 μV (p=0.66) respectively.

Figure 2:

Figure 2:

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Representative ERG waveforms obtained from light flashes of 10 cd s/m2 (a- and b-waves, top panel) representing a control (right) eye (black) and HI exposed (left) eyes with different waveform profiles. Exposed to 10 cd s/m2; mild injured eyes were almost completely normal (green), moderately injured eyes showed a slight reduction in b-wave amplitude (blue), and severely injured eyes showed a substantial decrease in both a- and b-wave amplitudes (red) in female mice (A-C). To further confirm HI affecting the inner retina, "oscillatory potentials" (OPs) originating in the inner retina were compared in different injury groups in female mice (D lower graphs).

Figure 3:

Figure 3:

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ERG waveforms obtained from 10 cd s/m2 light flashes (a- and b-waves, top panel) comparing a control (right) eye (black) to an HI exposed (left) eye in male mice. (B-C) Mildly injured eyes were normal (green), moderately injured eyes had a minor reduction in b-wave amplitude (blue), and severely injured eyes had an increase in a-wave and a decrease in b-wave amplitudes (red). Male mice' oscillatory potentials (OPs) are compared in different damage groups (D, lower graphs).

The third group of HI mice, here we termed as "severe," showed an increased a-wave only for males but not for the females, and both the females and males had a decreased b-wave. The injured female eyes had an a-wave of 220.23 ± 29.15 μV while the control eye had an a-wave of 210.16 ± 23.69 μV (p=0.79). The male a-wave for the injured eyes measured 190.6 ± 35.7 μV compared to 156.2 ± 35.79 μV (p=0.53) for the control eye. However, the b-wave for females reduced to 302.08 ± 49.73 μV compared to the control eye 472.78 ± 42.8 μV (p=0.02), and a similar result was obtained in injured male eye measuring 260.4 ± 29.07 μV compared to the control measuring 345 ± 66.5 μV (p=0.3) [Fig 2 and 3 (A-C)].

Riding on the b-wave response is a complex higher frequency oscillation termed "oscillatory potentials" (OPs) originating from the inner retina. We compared OP responses in these three groups to confirm HI effects in the inner retina. The mildly affected HI mice showed no difference in OPs in both eyes. We observed a slight reduction in amplitude in the affected eye compared to the control eye in the moderately affected HI mice. The OPs were significantly reduced in both eyes of severely affected mice, and the injured eye had an even smaller amplitude than the contralateral eye [Fig 2 and 3 (D)].

3.2. HI impairs percent changes in the b/a ratios of ERG in both sexes

The ERG amplitude recorded from the mice varies between mice. Given that we see a variation in b-wave amplitude and a minimum effect on the a-wave, we have taken a method to better categorize them based on the b/a ratio and assist better insight to assess the 7,8-DHF treated mice. A healthy eye response is noted as a larger b-wave compared to the a-wave response standardized by the International Society for Clinical Electroretinography of Vision (ISCEV), resulting in a b/a ratio of more than 2. Under pathological conditions, when the b-wave is smaller, the b/a ratio is less than 2, which is an electronegative response or negative ERG. Female and male control (right) eyes exhibited b/a ratios of 2.57 ± 0.05 and 2.55 ± 0.07, respectively. The b/a ratio result for the mild category female and male was more than 2 (Fig 4 A, B). The calculated b/a ratio in female mice was 2.11 ± 0.04 (p<0.05), and in males was 2.3 ± 0.07 (p<0.05). The female in the moderate category had the b/a values reduced to 1.82 ± 0.04 (p<0.05), and in males to 1.84 ± 0.02 (p<0.05). Both females and males with significantly reduced b-wave amplitude had the b/a ratio 1.2 ± 0.18 (p<0.05) and 1.12 ± 0.17 (p<0.05), respectively (Fig 4 A, B).

Figure 4:

Figure 4:

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The average plot of the b/a ratio of the control eye group compared to the three groups (mild, moderate, or severe groups) of HI injured eyes for both female (A) and male (B).

3.3. No sex differences in retinal function following neonatal HI and 7,8-DHF therapy in juvenile and young adult mice were detected

We have previously demonstrated that 7,8-DHF, a TrkB agonist/modulator, provides significant sex-specific neuroprotection in female hippocampi after neonatal HI (Uluc, Kendigelen et al. 2013, Cikla, Chanana et al. 2016). We performed ERG on mice at P30 to evaluate the effects of HI and 7,8-DHF treatment on the retinal functions stated in the methods section. The b/a ratio is calculated for both the injured (left) and the control (right) eye, and the injured/treated eye is compared to the contralateral eye to determine the extent of HI and treatment. Mice were subjected to electroretinogram (ERG) testing at P60 and P90 to examine and compare the long-term effects of HI and 7,8-DHF therapy. Compared to sham-operated mice, ERG indicated a considerable reduction in the b/a ratio following HI in both female and male mice. At P60 and P90, the ratio in the follow-up ERG for female mice continued to decline. The normalized values for the female mice at P30, P60, and P90 were 0.7 ± 0.02, 0.69 ± 0.02, and 0.64 ± 0.1 (p<0.05), respectively, indicating a 30% decrease in signal at P30 and P60, followed by a 36% decrease in signal at P90 (Fig 5A). In contrast, following HI, the normalized b/a ratio at P30 for male mice was 0.71 ± 0.05 (p<0.05). At P60 and P90, the normalized b/a ratio appears to have slightly improved, with values of 0.76 ± 0.05 and 0.78 ± 0.06 (p<0.05), respectively. At all three ERG periods, administration of 7,8-DHF did not improve the ERG waveform of male mice.

Figure 5:

Figure 5:

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At P9, the mice underwent either sham surgery or HI; the HI-T group also received 7,8-DHF treatment for 7 days. ERG was conducted at P30, P60, and P90, and the b/a ratio was calculated for both Females (A) and males (B) to see the effect of the treatment on both sexes.

P30, P60, and P90 remain comparable to those seen in untreated HI mice at 0.7 ± 0.07 (p=0.89), 0.63 ± 0.09 (p=0.27), and 0.74 ± 0.03 (p=0.65), respectively (Fig 5B). Interestingly, at each of the three ERG time points, female mice showed a slight but insignificant improvement in ERG findings with the values of 0.73 ± 0.03 (p=0.49), 0.74 ± 0.02 (p=0.12), and 0.8 ± 0.03 (p=0.21) at P30, P60, and P90, respectively (Fig 5A).

TrkB agonist/modulator therapy fails to recover % change in b/a ratios in both sexes (Figure 6). Although the percent change in the b/a ratios was statistically significantly increased compared to sham mice in both sexes post-HI, no statistical significance was found between the HI and HI-treated mice retina. These results suggest that neonatal HI deteriorates retinal function in both sexes in the juvenile and young adulthood periods.

Figure 6:

Figure 6:

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The mice were exposed to either sham surgery or HI at P9. 7,8-DHF treatment was given to the HI-T group for 7 days. ERG was performed at P30 (A, A'), P60 (B, B'), and P90 (C, C'). Percent (%) changes in the b/a ratio were calculated using the formula: 100 x [(right eye b/a ratio-left eye b/a ratio)/right eye b/a ratio]. There is no significant difference in % changes in b/a ratios between male sham, HI, and HI-T groups on P30 (Fig. A) and P60 (Fig. B). On P90 (Fig. C), there is a significant difference between the male sham and both HI and HI-T groups, while there is no significant difference. In female mice, shams are significantly different from the HI and HI-T groups on P30 (Fig. A'), different than the HI-T group in P60 (Fig. B'), and different than the HI group on P90 (Fig. C'). However, there is no significant difference between female HI and HI-T groups at any time.

3.4. Retinal function measured by % change in b/a ratio correlates significantly with the severity of injury in both sexes

T2 weighted MRI scans were obtained from sham, HI, and HI-treated mice at P90+ to quantify percent hemispheric tissue loss using ITK-SNAP. The percent hemispheric tissue loss positively correlates with the % change in the b/a ratio in adolescent and young adult mice of both sexes. At P30, P60, and P90, both male (Fig. 7 A, B, C) and female (Fig. 7 A', B', C') mice have a statistically significant correlation between their ERG and MRI results (A: p=0.01, A': p<0.0001; B: p=0.0077, B’=0.0190; C: p=0.0122, C': p<0.0001 respectively).

Figure 7:

Figure 7:

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The positive correlations between the % change in b/a ratio at ERG and %change in hemispheric volume loss at MRI are at P30, P60, and P90 in males (A, B, C) and in females (A', B', C') respectively. T2-Weighted MRI was performed under anesthesia at P90 using a Varian 4.7-tesla small animal MRI scanner (D). Masks of the hemispheric area were created for each slice using ITK-SNAP (E).

The percent hippocampal tissue loss has positively correlated with the % change in the b/a ratio in adolescent and young adult mice of both sexes. At P30, P60, and P90, both males (Fig. 8 A-C) and females (Fig. 8 A-C') mice have a statistically significant correlation between their ERG and MRI results (A: p=0.0061, A': p=0.0004; B: p=0.0080, B’=0.00130; C: p=0.0007, C': p=0.0001 respectively). When we look at the R2, which shows us the strength of the correlation, female mice have a higher R2 at P30 and P90. This might suggests that the ERG and MRI correlations in females are stronger than in males. These results indicate that ERG can be used as a noninvasive method for long-term prognostication of the severity of brain injury.

Figure 8:

Figure 8:

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The positive correlations between the % change in b/a ratio at ERG and %change in hippocampal volume loss at MRI are significant at P30, P60, and P90 in males (A, B, C) and females (A', B,' C') respectively. T2-Weighted MRI was performed under anesthesia at P90 using a Varian 4.7-tesla small animal MRI scanner (D). Masks of the hippocampal area were created for each slice using ITK-SNAP (E).

4. DISCUSSION

In this study, after inducing HI in male and female mice during the neonatal period, we tested the efficacy of TrkB agonist/modulator therapy called 7,8-DHF in the recovery of retinal function during adolescence and young adulthood. Our results show that HI selectively impairs inner retinal function by reducing the b-wave amplitude without altering the a-wave amplitude, which is consistent with other studies (Nilsson 1971, Jung, Polosa et al. 2015, Huang, Huang et al. 2021, Zaitoun and Sheibani 2021). Interestingly, the retinal function detected by ERG at each timepoint correlates strongly with the hemispheric and hippocampal brain injury obtained via brain MRI in both males and females, making ERG another very promising non-invasive method of detecting the severity of brain injury that can be translated to human neonates.

4.1. The damage to inner retina is selective and acute post-HI in both male and female mice and persists until adolescence and young adulthood

Human neonates with a diagnosis of HI-related brain injury, when tested with spectral-domain optical coherence tomography (OCT), showed a decrease in inner retina thickness and peripapillary nerve fiber impairment, which was found to be related to the severity of encephalopathy determined by the Sarnat criteria (Grego, Pignatto et al. 2021). Although clinical studies suggest that male neonates are more vulnerable to the effects of HI, the visual outcome of HI in animals has not been investigated in a sex-specific manner. In addition, bedside handheld OCT imaging within the first 2 weeks of life revealed the retinal injury in infants with HI-related brain injury (Mangalesh, et al., 2020). Other complications of neonatal HI, such as intraocular hemorrhage, have also been reported (Akin, et al. 2019). Although studies investigating the effect and implications of HI in the human term neonate’s visual system are scarce, emerging data from the preclinical animal studies demonstrate that the inner retina is more vulnerable to the effects of neonatal HI (Jung, et al. 2015, Huang, et al. 2018). In addition, using a mouse model of neonatal HI, aberrant retinal artery and vein sizes as well as a cease in the retinal vascular development, were observed (Zaitoun, et al. 2018, Zafer, et al. 2022).

In this study, we determined the HI effect on male and female retinas separately using ERG at P30, P60, and P90. Our results show that HI causes injury to the inner retina, which is comparable with previously mentioned studies performed in rats (Jung, Polosa et al. 2015, Huang, Huang et al. 2018). In addition, our results contribute to the literature by eliminating sex as a variable in the retinal damage of the mice exposed to HI during the neonatal period and tested during juvenile and early adulthood.

4.2. Sex-specific neuroprotective effects of TrkB agonist therapy do not extend to the retina

The TrkB signaling in neuronal survival, differentiation, migration, synaptic plasticity, and neuroprotection has been established in different preclinical injury models (Jiang, Wei et al. 2011, Uluc, Kendigelen et al. 2013, Xiang, Ren et al. 2015, Cikla, Chanana et al. 2016). Our lab previously reported a sexually differentiated mechanism post-HI in neonatal mice. TrkB agonist/modulator therapy with 7,8-DHF improved long-term hippocampal memory and learning assessment using the Morris Water Maze test only in female mice (Uluc, Kendigelen et al. 2013). However, the role of TrkB in sexually differential long-term neuroprotection of the retina post-HI is unknown. In a rat model of neonatal HI, when the first dose of 7,8-DHF treatment was administered before HI and repeated 18 hours after HI, rats showed survival of proliferating inner retinal cells, including Muller glia, and enhanced differentiation to bipolar cells at P17. Moreover, DHF treatment rescued the extracellular signal-regulated kinase (ERK) phosphorylation levels, which were significantly decreased after injury (Huang, Huang et al. 2018). None of these studies included sex as a variable in their analysis.

Contrary to the previous studies, we have included sex as a variable, but we did not detect any retinal functional recovery 7,8-DHF therapy. This could be due to the differences in the experimental design related to the timing of 7,8-DHF administration. We have chosen this experimental design to imitate the human scenarios and given the first dose of the 7,8-DHF after the induction of the HI. Other studies in adult rodents that utilized glaucoma and retinal ischemia-reperfusion injury showed the beneficial effects of the 7,8-DHF in protecting inner retinal cells (Gupta, Chitranshi et al. 2022, Yu, Wang et al. 2022). Another study with optic nerve transection also showed the neuroprotective effect of 7,8-DHF treatment in adult rats (Gallego-Ortega, Vidal-Villegas et al. 2021). Thus the protective effect of 7,8-DHF, although present in a sexually differential manner in neonatal mice post-HI in the hippocampal-dependent tasks, the similar experimental design does not recover inner retinal function measured by ERG. These differences in the 7,8-DHF therapy outcomes may be related to the age of the injury, the mechanism of the injury, and the timing of the dosing of the 7,8-DHF.

4.3. ERG as a diagnostic tool for the noninvasive assessment of the severity of brain injury

MRI imaging is the standard of care for neonates diagnosed with HI and is an essential tool for assessing the severity of brain injury. Hypoxic ischemic lesions in term neonates with mild and moderate HI were predominant in subcortical and deep white matter, while severe HI also involved basal ganglia, hippocampus, and thalamus (Bobba, Malhotra et al. 2022). Higher HI severity was associated with lesions in the hippocampus, thalamus, and lentiform nucleus (Bobba, Malhotra et al. 2022). Thus, in this study, we have performed the MRI of the mice that went through ERG to determine the severity of brain injury at P90+ and correlate the severity of injury with the percent change in the hippocampal and hemispheric tissue loss. Recent studies showed that in vivo high-field MRI allows for noninvasive evaluation of early postnatal development in impaired rat eyes (Au, Kancherla et al. 2021). Although MRI is a well-known tool providing a structural assessment of injury, it has limited power to show the functional evaluation. On the other hand, ERG is a diagnostic test used to measure electrical activity generated by neuronal and non-neuronal cells in the retina in response to light stimulus. In this study, we investigate the potential usage of ERG for the noninvasive evaluation of retinal function and prediction of long-term neurodevelopmental outcomes in neonatal HI. The correlation of percent change in the b/a ratio in the ERG with changes in volume loss in MRI in both sexes at all time points suggests ERG's future potential as a noninvasive assessment method for predicting long-term outcomes of retinal functions.

5. LIMITATIONS OF THE STUDY

Neurological disorders such as stroke, multiple sclerosis, and migraine show sex differences in the incidence and severity of illness (Clayton 2016). The effect of sex steroids has been implicated in the pathogenesis of the sex differences seen in these neurological disorders. It is well known that sex steroids regulate not only the function of reproductive organs but also the neural and behavioral functions (McEwen and Alves 1999). Role of sex steroids, especially estradiol, has been studied in adult animal models of age-related macular degeneration, oxidative retinopathies and glaucoma. Intravitreal hormone replacement therapy in the form of estradiol significantly recovered light-induced retinal damage by preventing neuronal apoptosis in ovariectomized female rat retinas (Mo, Li et al. 2013) and via its antioxidative effect in ovariectomized female and male rat retinas (Wang, Wang et al. 2015). Eight-week-old female rats exposed to surgical menopause have accelerated retinal neurodegeneration and increased susceptibility to insults in retinal ganglion cells (Olakowska, Rodak et al. 2022), which suggests estrogen therapy as a future therapeutic strategy for the treatment of glaucoma. On the other hand, the neuroprotective effect of estrogen in two different animal models of oxidative stress-induced retinopathies is controversial. Suppose the primary target of oxidative stress is the outer retina, estrogen is neuroprotective. However, in conditions where the inner retina is the main site of oxidative damage, estrogen may potentiate the detrimental effect of oxidative stress on male rat retinas (Chaychi, Polosa et al. 2018). In our study, we neither measured the sex steroid content of the mice retina nor treated the mice with sex steroids to assess the recovery. In future studies, we will consider studying the effect of sex steroid hormones on the recovery of visual functions post-HI.

6. CONCLUSION

In conclusion, to the best of our knowledge, this is the first study to investigate HI retinal damage and 7,8-DHF therapy in neonatal male and female mice separately. Our findings lighten three critical gaps in the literature. Firstly, the clinically observed sex difference in neurodevelopmental outcomes of HI does not exist in the retinal functions. Secondly, treating animals with 7,8-DHF does not have a similar protective effect on the retinal function when assessed by ERG in mice, as previously reported in the brain. Lastly, ERG can be utilized as a non-invasive tool to predict injury severity when an MRI is unavailable or to complement MRI findings to determine the severity of the injury. These findings suggest further research is needed to develop therapeutic approaches to prevent and reverse inner retina dysfunction for juveniles and young adults who suffer from HI during the perinatal period.

HIGHLIGHTS.

  • Neonatal hypoxia-ischemia (HI) results in severe inner retina function in mice

  • The loss of inner retinal function detected post-HI in mice is sex-independent.

  • A mild protective effect of TrkB agonist/modulator therapy on inner retina functions was found.

  • ERG can be a non-invasive assessment method for predicting long-term imaging outcomes.

ACKNOWLEDGEMENTS

We thank Jules Panksepp, Ph.D. at the Waisman Center Rodent Models Core, Behavioral Testing Service, and Beth M. Rauch, MS from Small Animal Imaging and Radiotherapy Facility (SAIRF), for making this research possible.

All persons who have made substantial contributions to the work reported in the manuscript (e.g., technical help, writing and editing assistance, general support), but who do not meet the criteria for authorship, are named in the Acknowledgements and have given us their written permission to be named. If we have not included an Acknowledgements, then that indicates that we have not received substantial contributions from non-authors.

FUNDING

This work was supported by the Department of Pediatrics, School of Medicine and Public Health, McPherson Eye Research Institute RRF MD Matthews Research Professorship (BRP), the National Institute of Health (R01EY024995) (BRP), Department of Pediatrics Research & Development Grant (PC), NIH/NINDS R01 NS111021 (PC), NIH/NINDS K08 NS088563 (PC), NIH/NINDS 1K08NS078113 (PF), NIH Waisman Core Grant P50HD105353.

ABBREVIATIONS:

HI

Hypoxia-ischemia

TrkB

tyrosine receptor kinase B

DHF

7,8-dihydroxyflavone

ERG

electroretinogram

MRI

magnetic resonance imaging

Footnotes

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REFERENCES

  1. Au JM, Kancherla S, Hamade M, Mendoza M and Chan KC (2021). "In vivo MRI evaluation of early postnatal development in normal and impaired rat eyes." Sci Rep 11(1): 15513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bobba PS, Malhotra A, Sheth KN, Taylor SN, Ment LR and Payabvash S (2022). "Brain injury patterns in hypoxic ischemic encephalopathy of term neonates." J Neuroimaging. [DOI] [PubMed] [Google Scholar]
  3. Chaychi S, Polosa A, Chemtob S and Lachapelle P (2018). "Evaluating the neuroprotective effect of 17beta-estradiol in rodent models of oxidative retinopathies." Doc Ophthalmol 137(3): 151–168. [DOI] [PubMed] [Google Scholar]
  4. Chokron S, Kovarski K and Dutton GN (2021). "Cortical Visual Impairments and Learning Disabilities." Front Hum Neurosci 15: 713316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cikla U, Chanana V, Kintner DB, Udho E, Eickhoff J, Sun W, Marquez S, Covert L, Otles A, Shapiro RA, Ferrazzano P, Vemuganti R, Levine JE and Cengiz P (2016). "ERalpha Signaling Is Required for TrkB-Mediated Hippocampal Neuroprotection in Female Neonatal Mice after Hypoxic Ischemic Encephalopathy(1,2,3)." eNeuro 3(1). [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clayton JA (2016). "Sex influences in neurological disorders: case studies and perspectives." Dialogues Clin Neurosci 18(4): 357–360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gallego-Ortega A, Vidal-Villegas B, Norte-Munoz M, Salinas-Navarro M, Aviles-Trigueros M, Villegas-Perez MP and Vidal-Sanz M (2021). "7,8-Dihydroxiflavone Maintains Retinal Functionality and Protects Various Types of RGCs in Adult Rats with Optic Nerve Transection." Int J Mol Sci 22(21). [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Graham EM, Ruis KA, Hartman AL, Northington FJ and Fox HE (2008). "A systematic review of the role of intrapartum hypoxia-ischemia in the causation of neonatal encephalopathy." Am J Obstet Gynecol 199(6): 587–595. [DOI] [PubMed] [Google Scholar]
  9. Grego L, Pignatto S, Busolini E, Rassu N, Samassa F, Prosperi R, Pittini C, Cattarossi L and Lanzetta P (2021). "Spectral-domain OCT changes in retina and optic nerve in children with hypoxic-ischaemic encephalopathy." Graefes Arch Clin Exp Ophthalmol 259(5): 1343–1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gupta V, Chitranshi N, Gupta V, You Y, Rajput R, Paulo JA, Mirzaei M, van den Buuse M and Graham SL (2022). "TrkB Receptor Agonist 7,8 Dihydroxyflavone is Protective Against the Inner Retinal Deficits Induced by Experimental Glaucoma." Neuroscience 490: 36–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huang HM, Huang CC, Hung PL and Chang YC (2012). "Hypoxic-ischemic retinal injury in rat pups." Pediatr Res 72(3): 224–231. [DOI] [PubMed] [Google Scholar]
  12. Huang HM, Huang CC, Poon LY and Chang YC (2021). "Artemin Is Upregulated by TrkB Agonist and Protects the Immature Retina Against Hypoxic-Ischemic Injury by Suppressing Neuroinflammation and Astrogliosis." Front Mol Neurosci 14: 645000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang HM, Huang CC, Tsai MH, Poon YC and Chang YC (2018). "Systemic 7,8-Dihydroxyflavone Treatment Protects Immature Retinas Against Hypoxic-Ischemic Injury via Muller Glia Regeneration and MAPK/ERK Activation." Invest Ophthalmol Vis Sci 59(7): 3124–3135. [DOI] [PubMed] [Google Scholar]
  14. Jang SW, Liu X, Yepes M, Shepherd KR, Miller GW, Liu Y, Wilson WD, Xiao G, Blanchi B, Sun YE and Ye K (2010). "A selective TrkB agonist with potent neurotrophic activities by 7,8-dihydroxyflavone." Proc Natl Acad Sci U S A 107(6): 2687–2692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jiang Y, Wei N, Lu T, Zhu J, Xu G and Liu X (2011). "Intranasal brain-derived neurotrophic factor protects brain from ischemic insult via modulating local inflammation in rats." Neuroscience 172: 398–405. [DOI] [PubMed] [Google Scholar]
  16. Jung S, Polosa A, Lachapelle P and Wintermark P (2015). "Visual Impairments Following Term Neonatal Encephalopathy: Do Retinal Impairments Also Play a Role?" Invest Ophthalmol Vis Sci 56(9): 5182–5193. [DOI] [PubMed] [Google Scholar]
  17. Kinoshita J and Peachey NS (2018). "Noninvasive Electroretinographic Procedures for the Study of the Mouse Retina." Curr Protoc Mouse Biol 8(1): 1–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Manjón JV, Coupé P, Martí-Bonmatí L, Collins DL and Robles M (2010). "Adaptive non-local means denoising of MR images with spatially varying noise levels." J Magn Reson Imaging 31(1): 192–203. [DOI] [PubMed] [Google Scholar]
  19. McEwen BS and Alves SE (1999). "Estrogen actions in the central nervous system." Endocr Rev 20(3): 279–307. [DOI] [PubMed] [Google Scholar]
  20. Mercuri E, Haataja L, Guzzetta A, Anker S, Cowan F, Rutherford M, Andrew R, Braddick O, Cioni G, Dubowitz L and Atkinson J (1999). "Visual function in term infants with hypoxic-ischaemic insults: correlation with neurodevelopment at 2 years of age." Arch Dis Child Fetal Neonatal Ed 80(2): F99–104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mo MS, Li HB, Wang BY, Wang SL, Zhu ZL and Yu XR (2013). "PI3K/Akt and NF-kappaB activation following intravitreal administration of 17beta-estradiol: neuroprotection of the rat retina from light-induced apoptosis." Neuroscience 228: 1–12. [DOI] [PubMed] [Google Scholar]
  22. Nilsson SE (1971). "Human retinal vascular obstructions. A quantitative correlation of angiographic and electroretinographic findings." Acta Ophthalmol (Copenh) 49(1): 111–133. [PubMed] [Google Scholar]
  23. Olakowska E, Rodak P, Pacwa A, Machowicz J, Machna B, Lewin-Kowalik J and Smedowski A (2022). "Surgical Menopause Impairs Retinal Conductivity and Worsens Prognosis in an Acute Model of Rat Optic Neuropathy." Cells 11(19). [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ozaydin B, Bicki E, Taparli OE, Sheikh TZ, Schmidt DK, Yapici S, Hackett MB, Karahan-Keles N, Eickhoff JC, Corcoran K, Lagoa-Miguel C, Guerrero Gonzalez J, Dean Iii DC, Sousa AMM, Ferrazzano PA, Levine JE and Cengiz P (2022). "Novel injury scoring tool for assessing brain injury following neonatal hypoxia-ischemia in mice." Dev Neurosci. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pinto LH, Invergo B, Shimomura K, Takahashi JS and Troy JB (2007). "Interpretation of the mouse electroretinogram." Doc Ophthalmol 115(3): 127–136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robson AG, Frishman LJ, Grigg J, Hamilton R, Jeffrey BG, Kondo M, Li S and McCulloch DL (2022). "ISCEV Standard for full-field clinical electroretinography (2022 update)." Doc Ophthalmol 144(3): 165–177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tustison NJ, Avants BB, Cook PA, Zheng Y, Egan A, Yushkevich PA and Gee JC (2010). "N4ITK: improved N3 bias correction." IEEE Trans Med Imaging 29(6): 1310–1320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tustison NJ, Cook PA, Holbrook AJ, Johnson HJ, Muschelli J, Devenyi GA, Duda JT, Das SR, Cullen NC, Gillen DL, Yassa MA, Stone JR, Gee JC and Avants BB (2021). "The ANTsX ecosystem for quantitative biological and medical imaging." Scientific Reports 11(1): 9068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Uluc K, Kendigelen P, Fidan E, Zhang L, Chanana V, Kintner D, Akture E, Song C, Ye K, Sun D, Ferrazzano P and Cengiz P (2013). "TrkB receptor agonist 7, 8 dihydroxyflavone triggers profound gender-dependent neuroprotection in mice after perinatal hypoxia and ischemia." CNS Neurol Disord Drug Targets 12(3): 360–370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Volpe JJ (2008). "Neurology of the newborn E-book." [PubMed] [Google Scholar]
  31. Wang S, Wang B, Feng Y, Mo M, Du F, Li H and Yu X (2015). "17beta-estradiol ameliorates light-induced retinal damage in Sprague-Dawley rats by reducing oxidative stress." J Mol Neurosci 55(1): 141–151. [DOI] [PubMed] [Google Scholar]
  32. Wojcik-Gryciuk A, Gajewska-Wozniak O, Kordecka K, Boguszewski PM, Waleszczyk W and Skup M (2020). "Neuroprotection of Retinal Ganglion Cells with AAV2-BDNF Pretreatment Restoring Normal TrkB Receptor Protein Levels in Glaucoma." Int J Mol Sci 21(17). [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Xiang L, Ren Y, Cai H, Zhao W and Song Y (2015). "MicroRNA-132 aggravates epileptiform discharges via suppression of BDNF/TrkB signaling in cultured hippocampal neurons." Brain Res 1622: 484–495. [DOI] [PubMed] [Google Scholar]
  34. Yu A, Wang S, Xing Y, Han M and Shao K (2022). "7,8-Dihydroxyflavone alleviates apoptosis and inflammation induced by retinal ischemia-reperfusion injury via activating TrkB/Akt/NF-kB signaling pathway." Int J Med Sci 19(1): 13–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zaitoun IS, Cikla U, Zafer D, Udho E, Almomani R, Suscha A, Cengiz P, Sorenson CM and Sheibani N (2018). "Attenuation of Retinal Vascular Development in Neonatal Mice Subjected to Hypoxic-Ischemic Encephalopathy." Sci Rep 8(1): 9166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zaitoun IS and Sheibani N (2021). "Hypoxic-Ischemic Encephalopathy: Impact on Retinal Neurovascular Integrity and Function." J Ophthalmic Vis Res 16(3): 317–319. [DOI] [PMC free article] [PubMed] [Google Scholar]

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