Effects of inter-alpha inhibitor proteins on brain injury after exposure of neonatal rats to severe hypoxia-ischemia

Abstract

Hypoxic-ischemic (HI) brain injury is one of the most common neurological problems occurring in premature and full-term infants after perinatal complications. Hypothermia is the only treatment approved for HI encephalopathy in newborns. However, this treatment is only partially protective, cannot be used to treat premature infants, and has limited efficacy to treat severe HI encephalopathy. Inflammation contributes to the evolution of HI brain injury in neonates. Inter-alpha Inhibitor Proteins (IAIPs) are immunomodulatory proteins that have neuroprotective properties after exposure to moderate HI in neonatal rats. The objective of the current study was to determine the neuroprotective efficacy of treatment with IAIPs starting immediately after or with a delay of one hour after exposure to severe HI of 120 minutes duration. One hundred and forty-six 7-day-old rat pups were randomized to sham control, HI and immediate treatment with IAIPs (60 mg/kg) or placebo (PL), and sham, HI and delayed treatment with IAIPs or PL. IAIPs or PL were given at zero, 24, and 48 hours after HI or 1, 24 and 48 hours after HI. Total brain infarct volume was determined 72 hours after exposure to HI. Treatment with IAIPs immediately after HI decreased (P<0.05) infarct volumes by 58.0% and 44.5% in male and female neonatal rats, respectively. Delayed treatment with IAIPs after HI decreased (P<0.05) infarct volumes by 23.7% in male, but not in female rats. We conclude that IAIPs exert neuroprotective effects even after exposure to severe HI in neonatal rats and appear to exhibit some sex-related differential effects.

1. Introduction

Hypoxic-ischemic (HI) brain injury is a major cause of neonatal morbidity, mortality and long-term neurodevelopmental disabilities (Barrett et al., 2007; Cotten and Shankaran, 2010; Scafidi et al., 2009). Developmental disabilities, including cerebral palsy, neurosensory and motor impairment, seizures, and/or intellectual deficits, place a large burden on families and society (Centers for Disease and Prevention, 2004). Hypothermia is the only approved therapeutic intervention currently available to treat hypoxic ischemic encephalopathy (HIE) in newborn infants. However, this therapeutic modality is only partially protective (McAdams and Juul, 2016; Perrone et al., 2012; Shankaran, 2012; Shankaran et al., 2012), can only be used to treat full term infants who have been exposed to HIE (Shankaran, 2012; Shankaran et al., 2012) and cannot be used to treat premature infants with HI related brain injury. Although hypothermia has been shown to improve neurological outcomes in human neonates with moderate HIE, it has much more limited efficacy in infants exposed to severe HIE (Edwards et al., 2010; Xu et al., 2020). Studies in neonatal rodents would appear to confirm the findings in human infants suggesting that hypothermia has much less neuroprotective efficacy after exposure to severe rather than moderate HI related brain injury (Sabir et al., 2012; Wood et al., 2016). Consequently, alternative, and/or adjunctive therapies are urgently needed to treat or attenuate brain injury resulting from exposure to HI related insults in neonates. Thus far, pharmacological agents are not routinely available to treat newborn infants, who have been exposed to HIE or premature infants with HI related brain injury.

Inter-alpha Inhibitor Proteins (IAIPs) are family of structurally related, naturally occurring, novel anti-inflammatory immunomodulatory proteins that inhibit destructive serine proteases and pro-inflammatory cytokines (Garantziotis et al., 2007; Singh et al., 2010). They have received increasing attention because of their contribution to many disease states including HI related brain injury in adult and newborn subjects (Barrios-Anderson et al., 2019; Chen et al., 2019; Fries and Blom, 2000; Lim, 2013; Singh et al., 2010; Threlkeld et al., 2014; Yano et al., 2003). The major forms found in human plasma are Inter-alpha Inhibitor (IaI), which contains two heavy chains and a single light chain, and Pre-alpha Inhibitor (PaI), which consists of one heavy chain and one light chain (Potempa et al., 1989; Zhu et al., 2008). The light chain is termed bikunin and includes two protease inhibitor domains that lead to its function as a serine protease inhibitor (Potempa et al., 1989). The heavy chains play an important role in the stabilization and construction of extracellular matrix tissues and synergize with the activity of bikunin (Zhu et al., 2008).

In recent studies, we have shown that treatment of neonatal rodents with human plasma derived IAIPs decreases the quantity of infarcted brain in both male and female neonatal rats (Chen et al., 2019). In addition, IAIP treatment has been shown to reduce neuronal cell death and neutrophilic infiltration of the brain, and improve histopathological outcomes, neuronal plasticity, and complex behavioural outcomes in male neonatal rats (Barrios-Anderson et al., 2019; Chen et al., 2019; Gaudet et al., 2016; Threlkeld et al., 2014; Threlkeld et al., 2017). Our previous studies have examined the efficacy of IAIPs in attenuating HI related brain injury predominantly after exposure to HI of moderate severity, achieved by carotid ligation and 90 minutes of exposure to hypoxia. However, the potential capacity for IAIPs to attenuate brain injury after exposure to a severe degree of HI remains to be determined. This is of critical importance because treatment with hypothermia is not neuroprotective after exposure to severe HI in neonatal rats (Sabir et al., 2012).

Treatment of infants after exposure to HIE is often delayed either because the time of the onset of injury before birth is not known and/or because the treatment requires evaluation and stabilization after birth (Higgins et al., 2011; Natarajan et al., 2018). Hypothermic treatment of infants with HIE is most effective when started within six hours after birth (Gunn et al., 1997; Shankaran et al., 2012). Therefore, it is necessary to examine the efficacy of delayed treatment with IAIPs as therapeutic agents after exposure of neonatal subjects to severe HI, and for future consideration of the use of IAIPs as an adjunctive treatment to hypothermia. The lifespan of the human is much longer than that of rodents, and the trajectory of brain development is more rapid in rodents than in human infants (Andersen, 2003; Boxenbaum, 1982). Therefore, the time span of six hours for a human newborn is most likely much shorter in neonatal rodents, such that approximately one hour for a neonatal rodent is probably similar to six hours for the human newborn (Andersen, 2003).

Based upon the above considerations, the objective of the current study was to investigate the potential neuroprotective efficacy of systemic treatment with relatively high dose IAIPs given immediately or after a one-hour delay following exposure to severe HI in both male and female neonatal rats. Body weight, brain weight, and brain infarct volumes were determined in the present study because they are known to be influenced by the severity of injury in response to HI (Sawada et al., 2000; Terao et al., 2008). The Rice-Vannucci method was used to induce severe HI in neonatal rats on postnatal day (P) 7 by exposure to right carotid artery ligation and 8% oxygen for a duration of 2 hours. The brain of P7–10 rats is generally considered to be similar to the brain of near-term infants (Boxenbaum, 1982; Cowan, 1979; Dobbing and Sands, 1979; Semple et al., 2013).

2. Methods

The experimental procedures in this study were performed after obtaining approval from the Institutional Animal Care and Use Committees (IACUC) of Brown University and of Women & Infants Hospital of Rhode Island. All experimental procedures were carried out in accordance with the National Institutes of Health (NIH) guide for the care and use of laboratory animals.

2.1. Preparation of IAIPs

IAIPs were produced and purified as previously described in detail (Lim, 2013; Opal et al., 2011; Spasova et al., 2014). Briefly, IAIPs were extracted from fresh frozen human plasma (Rhode Island Blood Center, RI, USA). A scalable purification process was used to extract IAIPs with a monolithic anion-exchange chromatographic media (CIMmultusTM, BIA Separation, Ajdovščina, Slovenia), and an additional separation step using a proprietary synthetic chemical ligand affinity chromatographic media (Astrea Bioseparations, Cambridge, UK) was applied to obtain high yield, high purity (>90%), and biologically active human plasma-derived IAIPs. Eluted proteins were concentrated and buffer-exchanged using a tangential flow filtration device (Labscale TFF System, MilliporeSigma, Burlington, MA, USA) using Pellicon XL50 cartridge with 30 kDa cut-off Biomax membrane (MilliporeSigma, Burlington, MA, USA). Analyses of the purity and biological activity of IAIPs were performed using SDS-PAGE, Western immunoblot, protein assay, and competitive immunoassay. The biological activity was measured based on the ability of IAIPs to inhibit the hydrolysis of N-benzoyl-L-arginine-p-nitroaniline HCl (L-BAPNA, MilliporeSigma, St. Louis, MO, USA) by trypsin (Lim et al., 2005; Threlkeld et al., 2014). Inhibition was observed by a decrease in the rate of change in absorbance per minute at 405–410 nm wavelengths (Lim et al., 2005). Endotoxin in the purified products was monitored using a limulus amebocyte lysate endotoxin-based chromogenic test (Pierce Biotechnology, ThermoFisher Scientific, Waltham, MA, USA). The chromatographic equipment and containers/tubing were treated with 1 M NaOH to reduce or eliminate potential endotoxin contamination during the purification process.

2.2. Animal preparation, surgical procedures, experimental design, and necropsies

Pregnant Wistar rats at embryonic day 15 or 16 were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed in a temperature controlled, 12-hour light/dark-cycled facility with ad libitum access to food and water in the Animal Care Facility at Brown University. The dates upon which the rat pups were delivered were confirmed and designated as postnatal day zero (P0). Litters were culled to a maximum of 10 pups on P1 such that there were as close to equal numbers of male and female pups within each litter as possible to reduce intra-litter variability.

The Rice-Vannucci method was used to induce HI in the neonatal rats (Rice et al., 1981). At P7, the pups were randomly assigned to one of three groups: Sham-operated controls (Sham), HI placebo-treated (HI-PL), or HI IAIP-treated (HI-IAIP). Surgery was performed after anesthetic induction with 4% isoflurane and maintained with 2% isoflurane using a Vapomatic Anesthetic Vaporizer (Bickford Anesthesia Equipment, Wales Center, NY, USA). Rat pup sex was documented. The rat pups were placed in a supine position on a heated surgical platform to maintain body temperature at 36°C during surgery. A small vertical midline incision of 0.5–1 cm was made above the suprasternal notch and the right common carotid artery was isolated from the trachea and surrounding veins and nerves to specifically avoid the vagus nerve. Two sterilized 5–0 silk sutures were looped around the right common carotid artery and tied tightly with two double knots in the HI-PL and HI-IAIP groups. In the Sham group, an equivalent incision was made, but the right common carotid artery was not ligated. The incision was closed and disinfected with betadine and 70% ethanol. Each pup was marked with tail ink injections for identification (Neo-9, Animal Identification & Marking Systems, Inc., Hornell, NY, USA), and returned to the dam for 1.5–3 h before exposure to hypoxia.

We have previously shown that 90 minutes of hypoxia (8% oxygen plus 92% nitrogen) induces moderate to severe HI related brain injury in HI-PL treated male and female neonatal rats (Chen et al., 2019). Thus, in the present studies, we placed pups in a temperature-controlled oxygen/hypoxia (8% oxygen with 92% nitrogen) airtight chamber (Biospherix, Parish, NY, USA) for 120 minutes in order to induce severe brain damage in the neonatal rats (Fernandez-Lopez et al., 2007; Geddes et al., 2001; Vannucci et al., 1997; Vannucci and Hagberg, 2004). One non-ligated sentinel pup per litter had a rectal temperature probe inserted with an accuracy of 0.1°C (RET-4, Physitemp, Clifton, NJ, USA) to monitor body temperature before and during hypoxia. The sentinel pup was not further included in the experimental investigations because it has been shown that the stress experienced during the placement of the rectal probe alters the outcome of the HI related brain injury (Thoresen et al., 1996; Thoresen et al., 2009). In addition, rectal temperature has been shown to reflect brain temperature accurately in the HI rodent model (Dingley et al., 2006; Thoresen et al., 1996). The rectal temperature was recorded every 10 minutes during hypoxia and maintained at 36.0°C (Osredkar et al., 2014; Thoresen et al., 2009). Sham subjects were placed in a similar chamber and remained in room air for 120 minutes.

The experimental designs for our study are illustrated in Figure 1. After recovery from surgery and exposure to hypoxia for 2 hours, the immediate treatment group (Figure 1A) was given three intraperitoneal (I.P., open arrows) injections of 60 mg/kg of human plasma-derived IAIPs (ProThera Biologics, Inc., Providence, RI, USA) immediately (time zero), 24, and 48 hours after the termination of hypoxia. In the delayed treatment group (Figure 1B), the pups were treated with I.P. injections of IAIPs (60 mg/kg, open arrows) at 1, 24, and 48 hours after the termination of hypoxia. We have previously shown that treatment with 30 mg/kg of IAIPs attenuates the development of HI related brain injury after exposure to 90 minutes of hypoxia (Chen et al., 2019; Threlkeld et al., 2014). In the current study, we elected to utilize a larger dose of IAIPs (60 mg/kg) in an effort to attenuate the potentially more extensive brain injury resulting from severe HI. The Sham treated groups were given phosphate buffered saline (PBS) or IAIPs, and HI-PL treated groups were given equivalent I.P. injection volumes of placebo (PBS) administered as described above. Separate time-matched, Sham or HI-PL pups were included for both immediate and delayed treatment groups for the measurement of infarct volumes.

Figure 1.

(A) Schematic for immediate treatment with IAIPs. The pups were returned to the dams for 1.5 to 3 hours after right common carotid artery ligation. Thereafter, the pups were exposed to 8% oxygen with balanced nitrogen for 120 minutes at a constant temperature of 36 °C. Sixty mg/kg of human blood derived IAIPs or placebo were given intraperitoneally (I.P.) immediately (zero), 24 and 48 hours after termination of hypoxia. Body weight was measured before surgery, immediately (zero), 24, 48 and 72 hours after hypoxia. Seventy-two hours after hypoxia, necropsy was performed, and brain weight obtained. (B) Schematic for the delayed treatment with IAIPs. The procedures were the same as for the schematic in A except that IAIPs or placebo were given 1, 24 and 48 hours after the termination of hypoxia. Body weight was measured before surgery, 1, 24, 48 and 72 hours after hypoxia. Seventy-two hours after hypoxia, necropsy was performed, and brain weight obtained. The − / + signs indicate the time (hours) before/after the end of hypoxia (0 hour). IAIPs = Inter-Alpha Inhibitor proteins, PBS = phosphate buffered saline.

The Sham treated groups received IAIPs (60 mg/kg, n = 11) or an equivalent volume of PBS (n = 9) in the immediate treatment group, and IAIPs (n = 21) or PBS (n = 19) in the delayed treatment groups. Differences were not observed in the values for any of the measured variables between the IAIP Sham or PBS Sham treated groups in either immediate or delayed treatment groups. Therefore, the IAIP and PBS Sham treated groups were combined into a single Sham group in both the immediate and delayed study groups.

Each pup was weighed before surgery, at the time of each IAIP or placebo injection, and at 72 hours after HI (body weights, solid circles). The brains were collected 72 hours after hypoxia (Figure 1, stars). The pups were sedated with a mixture of ketamine (74 mg/kg, I.P.) and xylazine (4 mg/kg, I.P.). Brains were perfused with PBS and 4% paraformaldehyde (PFA) via cardiac puncture at a flow rate of 3 mL/min. Thereafter, the brains were removed, weighed, post-fixed in PFA for 24 hours, and stored in 30% sucrose in phosphate buffer (0.1 M) at 4°C before cryo-sectioning for infarct volume analysis (Chen et al., 2019; Zhang et al., 2004).

2.3. Brain sectioning

To measure the infarct volume of HI brain injury, the rat pup brains were cut into four or five 2 mm coronal sections using a brain slicer matrix (Zivic instruments, Pittsburgh, PA, USA), immersed in optimal cutting temperature (OCT) embedding medium (Tissue-Tek, Sakura Finetek, Torrance, CA, USA), and frozen in a metal beaker containing isopentane (MilliporeSigma) surrounded by crushed dry ice (Chen et al., 2019; Zhang et al., 2004). Five cryosections (20 μm) were obtained from each 2 mm section and mounted on gelatin-coated Superfrost™ Plus microscope slides (Fisherbrand™, Fisher Scientific International, Inc., Hampton, NH, USA) and stored at −80°C before cresyl violet staining.

2.4. Cresyl violet staining

Each 2 mm coronal brain section was further divided into histological sections for analysis. Every third cryosection from each 2 mm section was obtained for histological quantification in the immediate treatment group. On the other hand, the first cryosection of each 2 mm section was obtained for histological quantification in the delayed treatment group. Each of these sections was selected to be stained with cresyl violet for evaluation of HI injury and stored at −80°C until staining. The cryosections were removed from the −80°C freezer and air-dried overnight. After exposure to 1:1 absolute ethanol/chloroform mixture (1 mL per slide) for 20 minutes, the sections were stained with cresyl violet (0.1%, w/v, MilliporeSigma) for 4–5 minutes, and then rinsed in distilled water for 1 minutes. Thereafter, the sections were differentiated in 95% ethanol for 1–4 minutes, dehydrated in 100% ethanol, and 95% ethanol for 1–4 minutes each, cleared in xylene for 5 minutes twice, air-dried overnight, and mounted with Cytoseal™ XYL (Richard-Allan Scientific™, San Diego, CA, USA).

2.5. Infarct volume measurement

Images of the cresyl violet-stained brain sections were obtained using a Micropublisher 6 CCD Camera (Qimaging, Surrey, British Columbia, Canada) in order to measure the infarct volumes of the whole hemispheres and damaged areas of the brains in the sham, HI placebo-treated, and HI IAIP-treated groups. The resultant images were analyzed with ImageJ (NIH, Bethesda, MD, USA) without knowledge of the group assignment. The respective volumes were calculated from each measured area by multiplying the distance between the sections. The infarct volumes were calculated as a percent ratio of the damaged hemisphere as the ratio to the total contralateral hemisphere with correction for hemispheric edema, according to the following formula: infarct (%) = [1 - (total ipsilateral hemisphere – ipsilateral hemisphere damage)/total contralateral hemisphere)] x 100% (Li et al., 2010; Sawada et al., 2000; Swanson et al., 1990).

2.6. Statistical analyses

All results were expressed as mean ± standard deviation (SD). Two factor ANOVA for repeated measures was used to compare body weight gain over time between the treatment groups. To further describe statistically significant differences in body weight gain over time in the Sham, HI-PL and HI-IAIP groups, separate ANOVAs for repeated measures were used to compare the Sham and HI-PL, Sham and HI-IAIP, and HI-PL and HI-IAIP groups over time. The body weight gain over time was also compared between the subjects in the immediate and delayed IAIP treatment groups by separate ANOVAs for repeated measures. The brain weight and infarct volume results were normally distributed based on the Shapiro Wilk W normality test, and thus were analyzed with one-way ANOVA. Sex differences in body and brain weights, and infarct volume were analyzed with factorial ANOVA to analyze interactive effects of multiple categorical independent variables (sex vs. treatment). If a significant difference was detected by ANOVA, the Tukey’s honestly significant difference (HSD) test for multiple comparisons was used as a post-hoc test. The brain weights and the percent infarct volumes between the subjects in the immediate and delayed IAIP treatment groups were also compared by ANOVAs with the Tukey’s honestly significant difference (HSD) test for multiple comparisons used as a post-hoc test. The generalized extreme studentized deviate (ESD) test was used to detect outliers (Rosner, 1983; Sozda et al., 2011; You et al., 2016) using Excel software (Microsoft Corporation, Redmond, Washington, USA). Outliers were only apparent in the infarct volume analysis for the delayed treatment group. Two outliers were detected in each of the sham, HI placebo-treated, and HI IAIP-treated groups and, consequently, were eliminated from the analysis. Except for the generalized ESD test, all statistical analyses were done using the STATISTICA package (TIBCO Software Inc., Palo Alto, CA, USA), and a P < 0.05 was considered to indicate statistical significance.

3. Results

3.1. Body weight gain and brain weights after immediate treatment with IAIPs or placebo in neonatal rats exposed to severe HI

Figure 2A contains the body weight gain plotted as a percent (%) for the total cohort of males and females, and separately for the males and females in the Sham, and HI-PL and HI-IAIP groups treated immediately after exposure to severe HI. The increase in body weight over time was greater in the Sham than in the HI-PL (ANOVA, cohort: F (1, 34) = 69.51, n = 36, P < 0.001) and HI-IAIP (ANOVA, cohort: F(1, 41) = 24.59, n = 43, P < 0.001) groups. In addition, the body weight gain over time was greater in the HI-IAIP than in the HI-PL (ANOVA, cohort: F (1, 37) = 4.43, n = 39, P = 0.042) group.

Figure 2.

Body weight gain (%) and brain weights (g) of the Sham, HI-PL and HI-IAIP groups after immediate treatment with IAIPs or placebo in neonatal rats exposed to HI related brain injury. (A) Percent body weight gain on the y-axis plotted against study time in hours on the x-axis in the total cohort of males + females, males, and females. Body weight gain (%) was lower in the HI-PL and HI-IAIP total cohort compared with the Sham group during the study periods. Body weight gain (%) was greater in the HI-IAIP total cohort compared with the HI-PL group during the studies. Body weight gain (%) was lower in the HI-PL and HI-IAIP males than in the Sham group during the study periods. The body weight gain (%) was greater in the HI-IAIP compared with HI-PL males but not in the females. Sham: male n = 10, female n = 10; HI-PL: male n = 8, female n = 8; HI-IAIP: male n = 11, female n = 12. Values are mean ± SD. (B) Brain weights in the total cohort of males plus females, males, and females plotted as dot plots showing values as mean ± SD. Brain weights (g) were lower in the HI-PL and HI-IAIP cohort and in the males than in the Sham group, but not in the females. Sham: male n = 10, female n = 10; HI-PL: male n = 8, female n = 8; HI-IAIP: male n = 11, female n = 12. *P < 0.05.

The body weight gain over time was greater in the Sham than in the HI-PL males (ANOVA, male: F(1, 16) = 35.57, n = 18, P < 0.001) and also greater than in the HI-IAIP males (ANOVA, male: F(1, 19) = 5.98, n = 21, P = 0.024). The body weight gain over time was also greater in the HI-IAIP compared with the HI-PL males (ANOVA, male: F (1, 17) = 6.27, n = 19, P = 0.022). Similarly, increases in body weight gain over time were observed in the female neonatal rats. The weight gain over time was greater in the Sham than in the HI-PL (ANOVA, females: F(1, 16) = 32.86, n = 18, P < 0.001) and in the HI-IAIP females (ANOVA, females: F(1, 20) = 21.08, n = 22, P = 0.001). However, differences were not observed between the HI-PL and HI-IAIP female subjects (ANOVA, females: F (1, 18) = 0.237, n = 20, P = 0.631). Statistical differences between males and females were not observed (Factorial ANOVA, F (2, 230) = 2.02, n = 236, P = 0.134) in the Sham, HI-PL or HI-IAIP groups.

Figure 2B contains the brain weights in the Sham, HI-PL, and HI-IAIP groups plotted for the combined cohort of males and females, and separately for the males and females. The brain weight differed among Sham, HI-PL, and HI-IAIP in the total cohort (ANOVA, cohort F (2, 56) = 7.87, n = 59, P = 0.001). The brain weights were heavier in the Sham compared with the HI-PL (Tukey’s HSD, cohort: P = 0.005) and HI-IAIP (Tukey’s HSD, cohort: P = 0.002) groups. However, the brain weights did not differ (Tukey’s HSD, cohort: P = 1.000) between the HI-PL and HI-IAIP groups in the total cohort. Similarly, the brain weights differed (ANOVA, males: F (2, 26) = 13.09, n = 29, P = 0.001) among the Sham, HI-PL, and HI-IAIP groups in the males. The brain weights were heavier in the Sham compared with the HI-PL (Tukey’s HSD, males: P = 0.001) and HI-IAIP (Tukey’s HSD, males: P = 0.002) males. However, the brain weights did not differ (Tukey’s HSD, males: P = 0.865) between the HI-PL and HI-IAIP males. There were no differences (ANOVA, female: F (2, 27) = 1.53, n = 30, P = 0.234) in the brain weights among the Sham, HI-PL and HI-IAIP groups in the females. Sex differences were not observed (Factorial ANOVA, F (2, 53) = 0.81, n = 59, P = 0.450) in brain weights between the Sham, HI-PL- and HI-IAIP male and female neonatal rats.

3.2. Decreases in ipsilateral hemispheric infarct volume losses after immediate treatment with IAIPs in neonatal rats exposed to severe HI

Figure 3A contains representative cresyl violet images of the coronal brain sections from the Sham, HI-PL and HI-IAIP males (top row) and females (bottom row). The cresyl violet stains neurons by binding to Nissl bodies resulting in a purple stain. The cresyl violet stain labels the rough endoplasmic reticulum in neurons located in the perikaryon and dendrites (Barr and Bertram, 1951). Decreases in the amount of Nissl body staining in neurons is associated with neural loss or degeneration (Barr and Bertram, 1951). Inspection of the coronal images revealed that the HI-PL group exhibited increased ipsilateral hemispheric pallor compared with the Sham group in both the male and female neonatal rats. Furthermore, the HI-IAIP treated group demonstrated reduced pallor compared with the HI-PL group in both male and female neonatal rats exposed to severe HI.

Figure 3.

Infarct volume measurement after immediate treatment with IAIPs. (A) Representative images of brain sections stained with cresyl violet 72 hours after exposure to severe HI brain injury. The “I” designates the hemisphere ipsilateral to the carotid artery ligation. Scale bar = 3 mm. Compared to the HI-PL hemisphere, the damaged area in both males (top row) and females (bottom row) were reduced after immediate treatment with IAIPs. (B) Percent infarct volume plotted on the y-axis for the Sham, HI-PL, and HI-IAIP groups on the x-axis for the cohort, males, and females. Values are mean ± SD. *P < 0.05. Sham: male n = 9, female n = 9; HI-PL: male n = 6, female n = 7; HI-IAIP: male n = 11, female n = 12.

Quantitative analysis of the percent infarct volume loss confirmed the appearance of the brain sections that had been stained with cresyl violet of the neonatal rats exposed to severe HI (Figure 3B). The ipsilateral hemispheric infarct volumes were substantially larger (64.2 ± 12.3%) in the HI-PL of the entire cohort compared (Tukey’s HSD, cohort: P = 0.001) with the Sham group (2.5 ± 3.6%). Treatment with IAIPs significantly reduced (Tukey’s HSD, cohort: P = 0.001) the infarct volume from 64.2 ± 12.3% to 31.2 ± 30.6% for the entire cohort. Therefore, treatment with IAIPs reduced the infarct volumes by 51.3 ± 46.5% in the entire cohort even after exposure to severe HI in the neonatal rats. Similarly, the infarct volume was 69.5 ± 6.4% in the HI-PL males compared (Tukey’s HSD, males: P = 0.0001) with 2.0 ± 3.5% in the Sham group and was reduced (Tukey’s HSD, males: P = 0.005) to 29.2 ± 33.3% by treatment with IAIPs. Therefore, treatment with IAIPs reduced the infarct volumes by 58.0 ± 47.9% in the male neonatal rats after exposure to severe HI. The findings were similar for the females. The ipsilateral infarct volume was 59.6 ± 14.6% in the HI-PL and 3.0 ± 3.8% in the Sham females (Tukey’s HSD, females: P = 0.001) and reduced (Tukey’s HSD, females: P = 0.003) to 33.1 ± 29.1% by treatment with IAIPs immediately after exposure to severe HI. Thus, treatment with IAIPs reduced the infarct volumes by 44.5 ± 48.9% in the female neonatal rats after exposure to severe HI. These findings can be interpreted to suggest that treatment with IAIPs immediately after exposure to severe HI has the ability to attenuate brain injury in both male and female neonatal rats.

3.3. Body weight gain and brain weights after delayed treatment with IAIPs or placebo in neonatal rats exposed to severe HI

Figure 4A contains the body weight gain plotted as a percent (%) for the total cohort of males and females, and separately for the males and females in the groups treated with IAIPs one hour after exposure to severe HI. The increase in body weight over time was greater in the Sham than in the HI-PL (ANOVA, cohort: F(1, 62 ) = 52.80, n = 64, P < 0.001) and HI-IAIP (ANOVA, cohort: F(1, 61) = 30.84, n = 63, P < 0.001) groups. However, the body weight gain over time did not differ between the HI-PL and HI-IAIP groups (ANOVA, cohort: F (1, 45) = 0.58, n = 47, P = 0.45). The body weight gain over time was greater in the Sham than in the HI-PL (ANOVA, males: F(1, 29) = 14.55, n = 31, P = 0.001) and HI-IAIP (ANOVA, males: F(1, 28) = 5.00, n = 30, P= 0.034) males but did not differ between the HI-IAIP and HI-PL (ANOVA, males: F(1, 21) = 1.32, n = 23, P = 0.264) groups. Similarly, the body weight gain over time was greater in the Sham than in the HI-PL (ANOVA, female: F(1, 31) = 52.42, n = 33, P < 0.001) and HI-IAIP (ANOVA, female: F(1, 31) = 40.72, n = 33, P < 0.0001) female groups. However, differences were not observed between the HI-PL and HI-IAIP females (ANOVA, female: F (1, 22) = 0.001, n= 24, P = 0.98).

Figure 4.

Body weight gain (%) and brain weights (g) of the Sham, HI-PL and HI-IAIP groups after delayed treatment with IAIPs or placebo in neonatal rats exposed to HI related brain injury. (A) Percent body weight gain on the y-axis plotted against study time in hours on the x-axis for the cohort, males, and females. Body weight gain (%) was lower in the HI-PL and HI-IAIP total cohort, males and females compared with the Sham group during the study periods. Sham: male n = 19, female n = 21; HI-PL: male n = 12, female n = 12; HI-IAIP: male n = 11, female n = 12. Values are mean ± SD. (B) Brain weights of the cohort, males, and females plotted as dot plots showing values as mean ± SD. Brain weights (g) were lower in the HI-PL and HI-IAIP cohort and in the HI-PL males, and in the HI-PL and HI-IAIP females compared with the Sham group. Sham: male n = 19, female n = 21; HI-PL: male n = 12, female n = 12; HI-IAIP: male n = 11, female n = 12. *P < 0.05.

The increase in body weight over time was greater in the immediate than in the delayed IAIP treatment groups for the total cohort of males and females (ANOVA, cohort: F (1, 182) = 5.48, n = 184, P = 0.020) and for the females (ANOVA, females: F (1, 94) = 10.41, n = 96, P = 0.002) but not for the males (ANOVA, males: F (1, 186) = 0.15, n = 88, P = 0.697).

Figure 4B contains the brain weights in the Sham, HI-PL and HI-IAIP groups plotted for the combined cohort of males and females, and separately for the males and females. The brain weight was heavier in the Sham compared with the HI-PL (Tukey’s HSD, cohort: P = 0.0002) and HI-IAIP (Tukey’s HSD, cohort: P = 0.005) groups. However, brain weights did not differ (Tukey’s HSD, P = 0.509) between the HI-PL and HI-IAIP total cohort groups. The brain weights were heavier (Tukey’s HSD, male: P = 0.016) in the Sham than in the HI-PL males, but did not differ (Tukey’s HSD, males: P = 0.414) between the Sham and HI-IAIP or between the HI-PL and HI-IAIP males (Tukey’s HSD, males: P = 0.344). The brain weights were heavier in the Sham than in the HI-PL (Tukey’s HSD, female: P = 0.002) and HI-IAIP (Tukey’s HSD, female: P = 0.004) females, but did not differ (Tukey’s HSD, females: P = 0.976) between the HI-PL and HI-IAIP females.

The brain weights did not differ between the subjects in the immediate or delayed IAIP treatment groups after exposure to severe HI in the total cohort (ANOVA, cohort: F (1, 44) = 0.46, n = 46, P = 0.502), in the males (ANOVA, males: F (1, 20) = 0.162, n = 22, P = 0.692) or in the females (ANOVA, females: F (1, 22) = 1.89, n = 24, P = 0.183).

3.4. Decreases in ipsilateral hemispheric infarct volume losses after delayed treatment with IAIPs in male but not in female neonatal rats exposed to severe HI

Figure 5A contains representative cresyl violet images of the coronal brain sections from the Sham, HI-PL and HI-IAIP males (top row) and females (bottom row) in the neonatal rats that were treated with placebo or IAIPs 1 hour after exposure to severe HI. Inspection of the coronal images reveal that the HI-PL group exhibited increased ipsilateral hemispheric pallor compared with the Sham group in both the male and female neonatal rats. Furthermore, the HI-IAIP treated group demonstrated reduced pallor compared with the HI-PL group in the male but not in female neonatal rats that had been exposed to severe HI.

Figure 5.

Infarct volume measurement after delayed IAIP treatment. (A) Representative images of brain sections stained with cresyl violet three days after HI brain injury for males (top row) and females (bottom row). The “I” designates the hemisphere ipsilateral to the carotid artery ligation. Scale bar = 3 mm. Compared to the HI-PL hemisphere, the infarct area in males was reduced after delayed IAIP treatment. (B) Percent infarct plotted on the y-axis for the Sham, HI-PL, and HI-IAIP groups on the x-axis for cohort, males, and females. Values are mean ± SD. *P < 0.05. Sham: male n = 18, female n = 19; HI-PL: male n = 10, female n = 12; HI-IAIP: male n = 10, female n = 11.

Quantitative analysis of the percent infarct volume loss confirmed the appearance of the cresyl violet stained images in the neonatal rats exposed to severe HI after delayed treatment with IAIPs (Figure 5B). The ipsilateral hemispheric infarct volumes were larger (45.4 ± 22.5%) in the HI-PL compared (Tukey’s HSD, cohort: P = 0.001) with the Sham (4.8 ± 3.0%) group for the entire cohort. Treatment with IAIPs did not reduce (Tukey’s HSD, P = 0.999) the infarct volumes in the entire cohort. The infarct volume was 62.5 ± 14.5% in the HI-PL males (Tukey’s HSD, males: P = 0.001), 3.6 ± 2.3% in the Sham group, and was reduced (Tukey’s HSD, males: P = 0.001) to 47.7 ± 20.1% by treatment with IAIPs one hour after exposure to severe HI. Therefore, treatment with IAIPs one hour after exposure to HI reduced the infarct volumes by 23.7 ± 32.1% in the males. In contrast, the ipsilateral infarct volumes were 33.9 ± 18.3% in the HI-PL and 5.9 ± 2.3% in the Sham females (Tukey’s HSD, females: P = 0.005) but the HI-related infarct volumes were not reduced (Tukey’s HSD, females: P = 0.231) by treatment with IAIPs one hour after exposure to severe HI in the female subjects. These findings can be interpreted to suggest that treatment with IAIPs one hour after exposure to severe HI reduces brain injury in male but not in female neonatal rats.

The ipsilateral hemispheric infarct volumes were larger in the delayed than in the immediate IAIP treatment group for the females (ANOVA, females: F (1, 21) = 1.41, n = 23, P = 0.025) but not for the total cohort (ANOVA, cohort: F (1, 42) = 3.84, n = 44, P = 0.0566) or for the males (ANOVA, males: F (1, 19) = 2.30, n = 21, P = 0.1456).

4. Discussion

The main objective of the current study was to determine whether treatment with human blood derived IAIPs reduces neuropathological brain injury characterized by the percent of total brain infarct volumes after exposure to an episode of severe HI in neonatal rats. The major finding of our study was that treatment with relatively high doses of human blood derived IAIPs attenuated neuropathologically determined brain volume loss when administered immediately after exposure to severe HI in both male and female neonatal rats. Furthermore, delayed treatment with the same doses of IAIPs attenuated the quantity of the brain volume loss in the male but not in the female neonatal rats.

The only therapy approved by the Federal Food and Drug Administration to treat newborn infants exposed to HIE is hypothermia (Shankaran, 2012). This treatment has a narrow therapeutic window and is most effective in the treatment of full-term infants that have experienced moderate rather than severe degrees of HIE (Edwards et al., 2010; Shankaran, 2012; Wood et al., 2016). Studies in neonatal rodents support the contention that hypothermia is more effective in the treatment of brain injury resulting from exposure to moderate rather than severe HI (Sabir et al., 2012). There are as yet no effective therapeutic pharmacological agents currently available to treat HIE in newborn infants.

In our previous report, we demonstrated that treatment with IAIPs at a dose of 30 mg/kg after exposure to HI induced by carotid artery ligation and 90 minutes of hypoxia reduced infarct volume loss in neonatal rats (Chen et al., 2019). Severity of brain injury is accentuated by increasing durations of exposure to HI in neonatal rats (Calvert et al., 2002; Vannucci et al., 1997; Vannucci and Hagberg, 2004). Carotid artery ligation and exposure to hypoxia for 90 minutes have previously been considered to be moderate HI (Sabir et al., 2012). In the current study, we administered 60 mg/kg of IAIPs immediately, 24 and 48 hours after exposure to HI resulting from carotid ligation and hypoxia for 120 minutes. The higher doses of IAIPs were selected because the larger doses might be more likely to attenuate brain injury after prolonged exposure to hypoxia (Calvert et al., 2002; Vannucci et al., 1997; Vannucci and Hagberg, 2004). Treatment with the 60 mg/kg doses of IAIPs immediately after exposure to HI attenuated the quantity of injury in both the males and female neonatal rats. Treatment with high dose IAIPs reduced the infarct volumes by 51.3 percent compared to our former work, in which the lower doses reduced the infarct volumes by 34.7 percent (Chen et al., 2019). However, additional research is required to determine whether the higher doses are actually more efficacious to attenuate brain injury than the lower doses in a study specifically designed to compare the effects of different doses under identical experimental conditions.

The decreases in the infarct volumes after treatment with IAIPs in the neonatal rats exposed to severe HI is particularly important because therapeutic hypothermia in the same rodent model was shown to be neuroprotective after exposure to moderate, but not to severe HI (Sabir et al., 2012). In our study, the neonatal rats were exposed to 120 minutes of 8% oxygen, compared with the previous report that examined the neuroprotective effects of hypothermia in rats exposed to hypoxia for 150 minutes (Sabir et al., 2012). However, the amount of brain injury appeared relatively comparable between the two studies because the rats in our study exposed to severe HI had infarct volumes of 64.2 percent compared with the 57 percent loss of area in the normothermic immediate treatment group in the previous study (Sabir et al., 2012), suggesting that the amount of HI related brain injury was similar between the two studies. Consequently, the findings of the current study, along with our former work (Barrios-Anderson et al., 2019; Chen et al., 2019; Threlkeld et al., 2014), can be interpreted to suggest that IAIPs could represent potentially important neuroprotective agents that could serve as alternative or adjunctive treatments to hypothermia.

The timing of treatment of HIE is often delayed in newborns infants because the timing of injury before birth is not known, delayed transfer of infants to an intensive care unit, in which therapeutic hypothermia can be performed, and/or because of the time required for stabilization and evaluation of the infant after exposure to HIE (Edwards et al., 2010; Natarajan et al., 2018). Clinical trials suggest that therapeutic hypothermia is most effective when the treatment is initiated within 6 hours of birth (Natarajan et al., 2018; Shankaran et al., 2012). However, infants are often enrolled for cooling within 3–4 hours after birth in recent trials (Natarajan et al., 2018). In our previous study, we examined the effects of delayed treatment with IAIPs (30 mg/kg) administered 6 hours after exposure to HI (Chen et al., 2019) and did not demonstrate that delayed treatment with IAIPs could reduce the average pathological scores across the brain regions in the male or female neonatal rats (Chen et al., 2019). In that study, the delay in IAIP treatment was 6 hours based upon the recommended time window after birth, in which therapeutic hypothermia is considered to be effective (Gunn et al., 1997; Gunn and Thoresen, 2019; Shankaran et al., 2012). However, the life span of a rodent is considerably shorter compared with the human (Boxenbaum, 1982). Therefore, a unit of time in the rat is approximately five times faster compared with humans based upon interspecies scaling between humans and rats (Boxenbaum, 1982). Consequently, a duration of 6 hours for a human would be approximately equivalent to 1.2 hours for a rat. Hence, in the current study, the neonatal rats were treated with IAIPs 1 hour after exposure to severe HI. Treatment with IAIPs 1 hour after exposure to severe HI reduced the infarct volume by 47.7 percent in the male but not in the female neonatal rats.

Reductions in infarct volumes were not detected in the female neonatal rats after the delayed treatment with IAIPs. It is important to emphasize that the infarct volumes in the placebo treated HI-PL male rats treated immediately and 1 hour after severe HI were similar, 69.5 and 62.5 percent, respectively. On the other hand, the values in the female rats were 59.6 and only 33.9 percent in the immediate and delayed HI-PL groups, respectively. It is well know that the Rice-Vannucci model of HI exhibits considerable variability (Failor et al., 2010; McQuillen and Ferriero, 2004; Sabir et al., 2012; Towfighi et al., 1997) and that female neonatal rats are often more resistant to injury compared with their male counterparts (Cohen and Stonestreet, 2014; Smith et al., 2014). Consequently, it is not surprising that we did not observe a decrease in the infarct volumes of the IAIP treated female rats.

The trajectory of weight gain during the studies after immediate treatment with IAIPs were similar to those in our previous report (Chen et al., 2019). The Sham, HI-PL and HI-IAIP groups each gained weight during the studies. The trajectory of weight gain was greater for the entire cohort and the male neonatal rats that were treated with IAIPs compared with the placebo treated HI-PL group. However, differences were not observed between the placebo and IAIP treated groups of females. The findings in the delayed treated group were also somewhat similar. Although significant differences in weight gain were not observed between the HI-PL and HI-IAIPs groups exposed to delayed treatment with IAIPs, we have previously shown that treatment with IAIPs attenuated HI related reductions in brain weight to some extent (Chen et al., 2019; Threlkeld et al., 2014). However, the reductions in brain weights were not spared by treatment with IAIPs in the immediate or delayed treatment groups after exposure to severe HI. The lack of attenuation in brain weight loss by treatment with IAIPs could be related to the longer exposure to hypoxia in the current study.

Comparisons between the groups treated immediately and after the one-hour delay in IAIP treatment did not exhibit significant differences in the brain weights in the total cohort, males, or females. Consistent with findings that the delayed treatment with IAIPs did not reduce the infarct the volumes in the female rats, immediate treatment resulted in greater reductions in the hemispheric infarct volumes compared with delayed treatment with IAIPs in the females, but not in the total cohort or in the males. Although the weight gain over time did not differ in the males based upon the timing of treatment with IAIPs, the weight gain over time was greater in the total cohort and in the females after the immediate treatment, rather than delayed treatment suggesting some potentially beneficial effects of the earlier treatment on the weight gain in total cohort and in the females. The mechanism(s) underlying the improved weight gain after immediate treatment with IAIPs in the total cohort and in the female neonatal rats cannot be discerned by our study but possibilities include increased intake, improved energy balance, and/or decreased activity after the immediate treatment with IAIPs (Chen et al., 2019).

The mechanism(s) by which IAIPs reduced infarct volumes after exposure to severe HI remain to be determined. However, we have previously demonstrated several potential mechanisms by which IAIPs attenuate brain injury in neonatal rats exposed to 90 minutes of hypoxia, including attenuation of HI related cell death, decreases in white matter injury, and decreases in astrocytic and microglial expression, and MPO-positive neutrophilic infiltration (Chen et al., 2019; Threlkeld et al., 2014). We have also shown that IAIPs reduce inflammatory mediated increases in blood-brain barrier permeability and that neutrophils treated with IAIPs in vitro more easily pass through the artificial microcapillaries and are prevented from entrapment inside the capillaries, reduce reactive oxygen species (ROS) in a concentration-dependent fashion and exhibit reduced adhesion to vascular endothelial monolayers (Htwe et al., 2018; Logsdon et al., 2020). In addition, the light chain of IAIPs, also known as bikunin or ulinastatin, has been shown to have protective effects in a variety of adult animal models including ischemia-reperfusion related brain injury and cardiovascular arrest (Lv et al., 2020). Ulinastatin has also been shown to diminish ischemia related increases in blood-brain barrier permeability by decreasing the expression of matrix metallopeptidase 9 and attenuating decreases in tight junction proteins, to suppress ischemia related oxidative stress, to reduce brain injury by decreasing apoptosis and to attenuate multiple features of inflammatory pathways (Cho et al., 2017; Hu et al., 2018; Jiang et al., 2016; Koga et al., 2010; Li et al., 2018; Lv et al., 2020). However, the potential mechanisms by which IAIPs reduced the infarct volumes after exposure to severe HI in the neonatal rats in the current study requires further investigation.

There are several limitations to the current study and opportunities for additional experiments because cresyl violet staining only shows an estimate of neuronal losses. More specific immunohistochemical staining for neuronal, astrocytic, microglia and oligodendrocytic markers have not been performed in the current study after exposure to severe HI. However, we have previously shown that IAIPs reduce the effects of HI on these more specific markers of brain injury in the neonatal rats exposed to 90 minutes of HI (Barrios-Anderson et al., 2019). This is certainly an important area of investigation and will be pursued in future studies. It is also important to perform dose response studies in order to determine whether the higher doses of IAIPs are actually required to attenuate the effects of severe HI on brain injury in the neonatal rats. This is particularly important with regard to the potential translational efficacy of IAIPs for the eventual consideration of IAIPs for human trials of neuroprotection.

5. Conclusions

We conclude that treatment with relatively high doses of human blood derived IAIPs attenuated neuropathologically determined brain volume loss when administered immediately after exposure to severe HI in both male and female neonatal rats, and that delayed treatment with IAIPs after severe HI decreased brain volume loss in male, but not in female rats. Therefore, IAIPs exert neuroprotective effects even after exposure to severe HI in neonatal rats and appear to exhibit some sex-related differential effects.

Highlights.

• Exogenous inter-alpha inhibitor proteins (IAIPs) reduce severe hypoxic-ischemic injury

• Exogenous IAIPs decrease hypoxic-ischemic infarction volumes in males and females

• IAIP treatment after a 1 hour reduces hypoxic-ischemic infarction volumes in male rats

Abbreviations

HI

Hypoxia-Ischemia

IAIPs

Inter-alpha Inhibitor Proteins

PL

placebo