PRAXIS.

Manal T. Usmani, Vipan K. Parihar, Charles. L. Limoli

INTRODUCTION

In April of 1961, Yuri Gagarin was the first human to travel to outer space. Curiosity is what drives space research, and astronauts continue to go to space to uncover the mysteries of the galaxy. Travelling to deep space can expose astronauts to galactic cosmic rays (GCR) which consist high energy charged particles such as proton, helium, silicon, oxygen and titanium (NASA, 2012). Long term exposure to GCR can potentially be dangerous to humans. The Parihar and Limoli Lab, in which I am a Biology 199 student and undergraduate research assistant, has determined that space irradiation causes persistent cognitive defects, and significant reductions in dendritic complexity and spine density (Parihar, 2015; Parihar, 2016). Prior studies have demonstrated pronounced cognitive and behavioral decrements in males after charged particle irradiation and new preliminary data indicates that females are significantly more resistant to these adverse neurocognitive effects (Parihar, 2016). Currently about 30% percent of astronauts are women, and women make up almost half of the 2017 NASA astronaut class (Northon, 2017). NASA research has a gender gap, and not just among humans. In many studies, the animals used are overwhelmingly male which may significantly reduce the reliability of the research because the results obtained on male animals may not hold up for women (Rabin, 2014; NIH 2012). Hence, my project aims to reveal groundwork for determining the extent and persistence of radiation-induced deficits in cognition in males and females, and to determine if cognitive defects following irradiation are sex-specific.

BACKGROUND AND OBJECTIVE

Microglia cells act as the Central Nervous System’s first line of immunological defense. As a response to certain types of radiation, these cells can become inflamed as an immune response to protect the brain (Green, UCI webpage). Activated microglia can either exert the M1 “harmful” phenotype or the M2 “beneficial” phenotype. M1 activation is involved with the release of a certain group of cytokines, which are proteins that help with cell communication and response, that promote inflammation (Cherry, 2014). Specifically, the 1L-1, IL-12, IL-18, Tumor Necrosis Factor, and COX2 cytokines are released (Wang, 2015). Conversely, M2 activation leads to greater expression of anti-inflammatory cytokines such as CD68 (Wang, 2015).

Humans do have mechanisms to activate microglia-mediated neuroinflammation. However, there are observed differences in the extent of inflammation between the sexes in both the healthy population and in many diseases. Both male and female astronauts are sent to space and can be exposed to cosmic radiation, which leads to the question of whether or not there are differences in their responses in space too.

OUTCOMES AND PREDICTIONS

Earlier this year, the Limoli Lab investigated whether or not microglia-mediated neuroinflammation following exposure to a typical type of radiation found in space was influenced by sex. Mice all aged 6 months old were exposed to whole body 4He ions (400 MeV) at a NASA Space Radiation Laboratory (NSRL) at Brookhaven National. Behavioral testing was then performed on the mice to reveal information about the potential effects of the radiation on cognition of the male versus female mice. Experiments were chosen and designed to give mice tasks that are controlled by the hippocampus, medial prefrontal and entorhinal cortical function. Cognitive defects are evident and recognizable to the researcher because normal function of these regions allows effective discrimination between a novel object and a familiar object, or an object at a new location. However, if these regions are impaired, mice will not be able to discriminate between a familiar and new object, or object at novel location.

Specific Aim: Determine whether females are more resistant to the adverse neurocognitive effects of cosmic ray exposure than males and whether this is due, in part, to reduced levels of neuroinflammation and oxidative stress.

NEW AIM: effect of estrogen, quantify the estrogen receptor in hippocampus and blood

Approach: Male and female C57BL/6J middle-aged mice, 6-month old, irradiated with 5 or 30 cGy of a helium ion irradiation, with concurrent controls that are not irradiated (N=24 per sex per group). Novel object Recognition (NOR) and Object in Place task (OiP) tests are conducted to interrogate the hippocampal, medial prefrontal and entorhinal cortical function at 12 weeks following radiation. Brains are harvested at 14 weeks after irradiation for neuroinflammation endpoints.

Preliminary data:

These important findings were derived from the experiment designed to uncover potential differences in behavior between the sexes following exposure to charged particles. Six-month old mice exposed to whole body 4He ions (400 MeV) were subjected to open arena testing using tasks that interrogate hippocampal, medial prefrontal cortex (mPFC) and entorhinal cortical function.

Impairment in these brain regions manifests as an inability to discriminate a novel from a familiar object, or an object at a new location. To quantify preference or indifference for exploring novelty, a discrimination index was calculated. A positive score signifies a preference indicated by more time exploring the novel object, whereas a negative score signifies indifference, or more time exploring the familiar object.

In the NOR task where the preference for novelty was determined after exposure to a novel object for 5 minutes, male control mice did show a preference for the novel object. However, when male mice were irradiated with 5 or 30 cGy of 4He ions, this preference was significantly diminished (Fig 1, left). When female mice were exposed to the same test, no significant differences were found between control and irradiated groups. A comparison of male to female mice found no significant differences between control groups, while irradiated male mice exhibited significant impairments in NOR after doses of 0.5 cGy and 30 cGy (Fig 1, left).

After NOR testing, mice were tested for OiP memory which is dependent on intact hippocampal and prefrontal cortex function. As with prior tasks, both male and female control groups showed an equivalent preference for object at the new location, while irradiated male mice scored significantly lower than female mice at both 5 and 30 cGy doses of 4He ions (Fig 1, right). These data demonstrate that 4He ion irradiation results in persistent and sex dependent deficits in object and place memory. As seen in Figure 1, the male and female control mice have very similar discrimination indexes, with the females scoring slightly higher. Irradiated females scored similar to the counterpart female controls. In contrast, irradiated males show a significant impairment in object recognition memory compared to the control males.

Following completion of behavioral testing, all of the mice were first euthanized and then underwent profusion, in order to remove and fix the brain for analysis of neuroinflammation through immunostaining. Assessment of the entorhinal cortex of mice brains revealed that control female mice had more cells that were CD68 positive and COX2 negative, suggesting that they have more of the M2 “beneficial” type of microglia phenotype compared to control male mice. After females were irradiated, the total number of activated microglia (both M1 and M2) was unaffected compared to the female controls (Fig 2). Interestingly, the total number of activated microglia increased by a 3.5 fold in male mice compared to the male controls. Imaging the stained brains revealed that the male mice were expressing greater numbers of the M1 proinflammatory phenotype as seen in Figure 2, which may contribute to their enhanced inflammation and neurocognitive decline after irradiation.

Data that supports this idea is related to reduced oxidative stress and inflammation in female mice, which is responsible for their unique response to radiation in comparison to males. High mobility group box-1 (HMGB1) protein is an important proinflammatory mediator that can activate inflammation. HMGB1 was increased in the entorhinal cortex in male mice 6 weeks after irradiation as seen in Figure 3. Female mice did not show similar results (Fig 3). These findings indicate that there are definitely core differences between Central Nervous System radiation responses between males and females, especially having major impact on neurocognitive abilities of male mice.

The hypothesis derived from this preliminary data is that greater numbers of basal M2 microglia in females primes their brains for response to irradiation stress. This can possibly be explained by potential down regulation of proinflammatory pathways which suppress microglial polarization to the M1 phenotype and/or by promoting microglia polarization to the anti-inflammatory M2 phenotype.

PROJECT DESIGN AND APPROACH

Subjects

Male and female C57BL/6J middle-aged mice, 6-month old mice (N=24 per sex per group).

Single beam whole body 4He ions (400 MeV) ion irradiation, 5 or 30 cGy.

Methods and Materials

The methods followed will be similar to the preliminary experiment described. Mice will first undergo behavioral testing including NOR and OiP. Before these tests, however, mice will undergo Elevated Plus Maze (EPM) and Forced Swim Test (FST) that determine anxiety-like and depression-like behavior, respectively, that will perhaps support the results obtained in the NOR and OiP testing. All of these tests will be video recorded. These videos will be watched and scored for their respective purposes, and a discrimination index will be produced. After this, the mice will be euthanized and their brains will be isolated for cryosectioning. A cryostat will be used to section 30 µm thick slices of the brains that will be placed in well plates, posterior to anterior. Specific sections that include the hippocampus will be picked up from the well and stained through principles of immunohistochemistry with antibodies that will bind to specific receptors that will indicate whether or not cells are releasing proteins that promote inflammation.

Novel Object Recognition (NOR): Successful performance of NOR is dependent on intact medial prefrontal cortex and hippocampal function and assesses recognition memory via spontaneous novelty preference. Following two-day habituation, a familiarization phase will be administered in which mice will be placed in the arenas and allowed to explore freely for 5 minutes with two identical toy blocks. Animals are then placed back into their home cage while the objects are cleaned (70% ethanol), and for the placement of a novel object. Mice are then returned to the arena for 5 minutes, where they explore the familiar and novel object. As in all cases, ANOVA (a statistical analysis) will be used to compare the time exploring the novel object between the experimental groups.

Object-in-Place (OiP): This test is also dependent on intact hippocampal and perirhinal cortex function and interrogates associative and place recognition memory. Following NOR testing, mice will be subjected to the OiP task. Briefly, mice will be habituated in their arenas (10 min/day) for 2 consecutive days. On the third day, mice will be exposed to four objects of varying size, color, and shape for 5 min (familiarization). Mice are then returned to their home cage (5 min) while objects are cleaned, and for the relocation of two of the four objects that switch locations. Mice having intact cortical function will show a preference toward the objects that have been switched to a novel location. Again, ANOVA will be used to compare the time exploring the objects at familiar and novel locations between the experimental groups.

Feasibility and Student Familiarity

This project is definitely feasible since a similar version of it has been successfully completed throughout the months of this past year. In this project, additional data will be collected in different sets of cognitive testing and brain tissue analysis to confirm sex specific cognitive defects. I have been, and still am, training extensively since mid-September to successfully perform the procedures discussed in the methods section. I am confident that I can apply what I have learned from my mistakes and achievements throughout these past two months to this upcoming project with the help of my supervisor and lab staff.

SPECIFIC RESPONSIBILITIES

Conduct the behavior tests that determine anxiety-and-depression-like-behavior using Elevated Plus Maze (EPM) and Forced Swim Test (FST).

Determine Novel Object Recognition (NOR) and Object-in-place Memory (OiP) using open arena tests.

Isolation of brains after mice are perfused with saline or 4 % PFA.

Cryosectioning of the brain (30 µm sections).

Immunohistological analysis through staining that determines inflammation mediators and neurodegeneration.

Confocal imaging and MBF stereology.

Sources:

Cherry, Jonathan D, John A Olschowka, and M Kerry O’Banion. “Neuroinflammation and M2 Microglia: The Good, the Bad, and the Inflamed.” Journal of Neuroinflammation 11 (2014): 98. PMC. Web. 7 Nov. 2017.

“Galactic Cosmic Rays.” NASA, NASA, 11 May 2012, helios.gsfc.nasa.gov/gcr.html.

Green, Kim. “Microglial Function in the Healthy Brain.” The Green Lab, UC Irvine, faculty.sites.uci.edu/kimgreen/bio/microglia-in-the-healthy-brain/.

“NIH Director Announces Appointment of Janine Clayton as Director of the Office of Research on Women's Health (ORWH) and the Associate Director for Research on Women's Health.” National Institutes of Health, U.S. Department of Health and Human Services, 4 Sept. 2012, www.nih.gov/news-events/news-releases/nih-director-announces-appointment-janine-clayton-director-office-research-womens-health-orwh-associate-director-research-womens-health.

Northon, Karen. “NASA Announces Its 2017 Astronaut Candidates.” NASA, NASA, 3 Aug. 2017, www.nasa.gov/press-release/nasa-s-newest-astronaut-recruits-to-conduct-research-off-the-earth-for-the-earth-and.

Parihar VK, Allen BD, Caressi C, Kwok S, Chu E, Tran KK, Chmielewski NN, Giedzinski E, Acharya MM, Britten RA, Baulch JE and Limoli CL (2016). Cosmic radiation and persistent cognitive dysfunctions. Sci Rep. 6: 34774.

Parihar VK, Allen B, Tran KK, Macaraeg TG, Chu EM, Kwok SF, Chmielewski NN, Craver BM, Baulch JE, Acharya MM, Cucinotta FA, Limoli CL (2015). What happens to your brain on the way to Mars. Sci. Adv. 2015;1:e1400256.

Rabin, Roni Caryn. “Labs Are Told to Start Including a Neglected Variable: Females.” The New York Times, The New York Times, 14 May 2014, www.nytimes.com/2014/05/15/health/nih-tells-researchers-to-end-sex-bias-in-early-studies.html.

Wang, Wen-Ying et al. “Role of pro-Inflammatory Cytokines Released from Microglia in Alzheimer’s Disease.” Annals of Translational Medicine 3.10 (2015): 136. PMC. Web. 7 Nov. 2017.