Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD

Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a "superintervention" which combines wheel running and environmental complexity and will address the limitations of these interventions.

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, such as rates of adult neurogenesis, angiogenesis, and expression of neurotrophic factors in rodents. This intervention has also been shown to mitigate behavioral and neuroanatomical aspects of the negative impacts of teratogens (i.e., developmental exposure to alcohol) and age-related neurodegeneration in rodents. Environmental complexity (EC) has been shown to produce numerous neuroplastic benefits in cortical and subcortical structures and can be coupled with wheel running to increase the proliferation and survival of new cells in the adult hippocampus. The combination of these two interventions provides a robust "superintervention" (WR-EC) that can be implemented in a range of rodent models of neurological disorders. We will discuss the implementation of WR/EC and its constituent interventions for use as a more powerful therapeutic intervention in rats using the animal model of prenatal exposure to alcohol in humans. We will also discuss which elements of the procedures are absolutely necessary for the interventions and which ones may be altered depending on the experimenter's question or facilities.

Rearing in different environments has long been known to cause changes in various measures of neurological wellness. Many studies look at the beneficial effects of rearing in a complex environment (EC) starting with groundbreaking research by Diamond and Rosenzweig (e.g., ^1,2) and Greenough (e.g.,^3,4). EC has been demonstrated to have an undeniable positive effects on synaptic and cellular changes in the brain^5,6,7. EC can affect a multiplicity of brain regions including the hippocampus^8,9 and visual cortex^10,11, ventral striatum^12,13, as well as brain-wide neuroimmune function (reviewed in¹⁴). Particular interest has developed from the studies on hippocampus when it was demonstrated that EC can increase the survival rate of adult-born granule cells of the dentate gyrus through dendritic plasticity ^9,13. This last point has gathered much interest due to the growing body of literature indicating that cardiovascular exercise promotes adult neurogenesis in both the healthy and damaged brain^15,16,17,18. Wheel running (WR) is an easy to implement form of voluntary cardiovascular activity that has been shown to be beneficial in rodent models of neurological disorders or aging ^17,19,20. WR affects the expression of growth factors in both the central and peripheral nervous system ^21,22,23.

Combining (subsequently) WR and EC into a "superintervention" (WR-EC) (i.e., 12 days of WR followed by 30 days in EC) provides a robust increase in hippocampal adult neurogenesis and increased survival of the newly proliferated cells⁸, the effect that in the animal model of FASD is not achieved by individual components (see below). Since both components of WR-EC affect a diverse array of structures within the brain¹³ (WR reviewed in²², EC reviewed in²⁴), implementation of this intervention can easily be applied to rodent models of both developmental and later life onset models of neurological impairment (e.g., neonatal alcohol exposure, aging, early life stress).

Integration of WR-EC in the adolescent and early adult periods (i.e., postnatal days 30 - 72) can ameliorate some of the negative effects of a rat model of fetal alcohol spectrum disorders (FASDs)⁸. A collection of studies have demonstrated that rodents exposed to alcohol from postnatal day (PD) 4 through 9 display significant deficits in neuroanatomical measures such as dendritic complexity²⁵, cerebellar development^26,27 and neuroimmune responsiveness²⁸ as well as manifestations of impaired learning and memory^29,30,31. Even a reduced amount of alcohol exposure within this time window (i.e., PD 7 through 9) can lead to deficits in learning and memory in adolescent and adult rats³² while some structures no longer see significant neuroanatomical impairment²⁷. Many of these deficits - in addition to behavioral impairments in hippocampus-dependent tasks - have been mitigated following exposure to this WR-EC paradigm^8,33 or WR alone^25,31. Although WR alone has been a widely used intervention, the combination of WR-EC has not yet been utilized in the literature despite its ability to sustain the relatively shorter-term benefits of WR⁸. This article will discuss the implementation of the WR-EC intervention during adolescence. Although this paradigm is used in the context of early postnatal alcohol exposure, it can be introduced to various rodent models to assess brain potential for neuroplasticity in the models of brain disorders.

Ethics Statement: The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Delaware.

1. Developmental Exposure (or Model of Binge-like Ethanol Exposure)

1. On PD3, determine the sex of each animal and cross-foster any animals if necessary to keep litter size (8 animals) and sex distribution (4 males : 4 females) consistent within each litter.
   NOTE: It is important to keep litter size and sex distribution as consistent as possible to avoid experimental confounds. Although this protocol uses 8 pups (4 males and 4 females) per litter, alternative litter sizes or sex distributions may be tailored to the needs of the experimental design.
2. Subcutaneously inject a small amount of black India ink into the paws to identify animals within each litter.
3. Pseudo-randomly assign litters as experimental (containing 50% alcohol-exposed (AE) and 50% sham-intubated control (SI) pups) or suckle control (SC) (animals that do not undergo any intubation, tail clipping, or separation protocols from PD 4 - 9 except for daily weighing and ear-punching).
   1. To retain consistent group size, assign twice as many experimental litters as SC litters.
4. Weigh each animal then return it to its home cage. Animal weighing should occur daily during the intubation period (PD 4 - 9).
   1. Remove the whole litter from the dam.
   2. Place pups on heated pad.
   3. Record the weight of each individual pup.
5. On PD4, after weighing each animal calculate the necessary alcohol amount for a total of 5.25 g/kg/day per each animal (based on pup weight from step 1.4)⁸.
   1. Administer alcohol as 11.9% ethanol-in-milk substitute (vol/vol).
6. Starting at 9 AM, remove one litter's pups from the mother at a time.
7. Administer the ethanol-in-milk to each AE pup³⁴.
8. Sham-intubate each SI pup⁸.
9. Repeat steps 1.5. through 1.8. for each experimental litter.
10. Two hours following the first dose, repeat the dosing procedure (steps 1.5 through 1.8) for a second alcohol dose.
11. One and a half hours after the second alcohol dose (the point at which peak daily blood alcohol content is achieved), collect and centrifuge blood from the AE and SI pups via tail clipping for future blood alcohol content analysis³⁵.
    1. Collect 60 µL of blood.
    2. Place blood in a 1 mL microcentrifuge tube. Centrifuge blood at 1.5 x g for 25 min.
    3. Carefully collect the supernatant serum from the centrifuge tube and save for future blood alcohol content analysis.
12. Repeat the dosing procedure (steps 1.5 through 1.8) using milk instead of ethanol-in-milk to prevent nutritional deficits from nursing inability in AE pups.
    1. Perform a total of 2 supplemental milk doses 2 h apart on PD 4.
13. Repeat steps 1.4 through 1.12 (except for step 1.11) on PD 5 – 9.
14. Following the final supplemental milk dose on PD9, ear punch all pups for identification in the EC cage.
    1. Coordinate punched ear with some measure of litter number or identifier (e.g., odd numbered litters within a cohort would get their left ear punched while animals from even numbered litters would get their right ears punched). This will make it easier to identify animals in the EC cage should multiple animals from different litters have the same pawmark pattern.

2. Weaning

1. On PD 23, house all animals in cages of 2 - 3.
   1. Ensure that all animals housed in the same cage are the same sex.
   2. Include one SC, one SI, and one AE animal per cage when possible.
   3. Minimize the number of cage mates that are from the same litter.
   4. Make sure all animals are capable of accessing food and water.

3. Wheel Running

1. On PD30, allocate half of the cages with animals to WR. House these animals in cages with a free access to attached stainless steel running wheel.
   1. Ensure that wheels have a counter to assess the total number of revolutions.
2. Weigh all animals on PD 30 and PD 36.
3. Check the number of revolutions of each wheel at 9 AM every day.
4. Leave animals in their respective housing condition for 12 days.

4. Environmental Complexity

1. Prepare the EC cage before 9 AM on the day that corresponds to PD 42 for experimental animals.
   1. Get a 30 " x 18 " x 36 " galvanized steel cage.
      NOTE: The cage should have multiple levels, be capable of supporting the weight of multiple rats, be filled with standard bedding, and have multiple locations to attach water bottles and food dispensers.
   2. Place novel, colorful objects of variable sizes and shapes in the cage.
      1. Place 6 large toys in the EC cage. Ensure that each toy is big enough for 3 or more rats to interact with concurrently.
      2. Place 6 medium toys in the EC cage. Ensure that each toy is big enough for 3 - 4 rats to interact with concurrently.
      3. Place a lot (at least 20) of smaller toys in the EC cage.
      4. Use toys of varying colors, shapes, size, etc. Novelty is critical to this intervention (see discussion).
   3. Place two dishes of food at opposite ends of the cage.
   4. Place two bottles of water at opposite ends of the cage.
2. At 9 AM on PD 42, weigh all animals and relocate the WR animals to the EC cage. Each EC cage should contain 9 - 12 animals.
   1. Make sure that no animals have both the same pawmark and ear-punch patterns.
3. Check all food and water daily.
4. Every two days, remove the toys from the EC cage and replace them (according to step 4.1.2.).
5. Every three days, clean the EC cage.
   1. Remove the animals from the EC cage and put them in temporary holding cages of 2 - 3 animals.
   2. Remove all of the bedding from the bottom of the cage.
   3. Return the same toys to the cage unless this day coincides with the toy replacement schedule (according to step 4.4.).
   4. Replace all of the food and water.
   5. Replace the rats into the EC cage.

5. Collect Tissue

NOTE: Tissue collection (e.g., perfusion with paraformaldehyde) and storage (e.g., freezing, paraffin embedding) can be performed with a variety of methods. The following will explain the process of perfusion with 4% paraformaldehyde in 0.1 M phosphate buffered saline (4% paraformaldehyde in PBS) solution⁸.

Caution: Paraformaldehyde is carcinogenic and may also cause skin irritation, allergic skin reaction, or eye damage. Use appropriate eye/skin protection.

1. Expose one rat at a time to isoflurane to lightly anesthetize the animal.
2. Intraperitoneally inject the rat with 2 mL/kg of Ketamine/xylazine mixture (1.5 mL xylazine mixed with 10 mL of ketamine).
   NOTE: Ketamine and xylazine are both at stock concentrations of 100 mg/mL before combining for injection mixture.
3. Once rat is no longer responsive, perfuse the animal with 0.1 M phosphate buffered saline (PBS; pH = 7.2) followed by 4% paraformaldehyde in PBS (pH = 7.2).
4. Remove brain and store in 4% paraformaldehyde in PBS at 4 °C for 48 h.
5. After 2 days, transfer to solution of 30% sucrose added to 4% paraformaldehyde in PBS at 4 °C.

In order to assess the effect of the super intervention, we must look at the effects of each of its constituent elements — WR and EC — on our measures of interest. Figures 1 through 3 (below) appeared in a previous publication utilizing this paradigm⁸. Figure 4 appeared in a doctoral dissertation³⁶. These data illustrate the impact of WR-EC on hippocampal adult neurogenesis in th...

In the above protocol, we demonstrated an expedient intervention to rescue neuroanatomical deficits following neonatal alcohol exposure. This intervention can be used as a therapeutic in other animal models due to the robustness of each of the components of the intervention. Voluntary cardiovascular activity in the form of WR has been shown to benefit several behavioral outcomes^38,39 and induce functional plastic alterations in brain regions such as the hippocamp...

The authors have nothing to disclose.

We would like to dedicate this work to the memory of late Dr. William T. Greenough, a great mentor, a colleague and a friend. This work was supported by NIH/NIAAA grant number AA009838 and NIH/NIGMS COBRE: The Delaware Center for Neuroscience research grant 1P20GM103653 to AYK. We are grateful to the former and current members of Klintsova lab.