Brain extracellular space of the naked mole-rat expands and maintains normal diffusion under ischemic conditions

Highlights

• Brain extracellular space (ECS) of African naked mole-rats was assessed in vitro.

• ECS volume and diffusion of macromolecules were lower than typical rat values.

• Thick cortical slices paradoxically increased ECS volume and preserved diffusion.

• Extracellular potassium accumulation was blunted in the thick slice preparation.

Abstract

Brain extracellular space (ECS) forms a conduit for diffusion, an essential mode of molecular transport between brain vasculature, neurons and glia. ECS volume is reduced under conditions of hypoxia and ischemia, contributing to impaired extracellular diffusion and consequent neuronal dysfunction and death. We investigated the ECS volume fraction and diffusion permeability of the African naked mole-rat (NM-R; Heterocephalus Glaber), a rodent with a remarkably high tolerance for hypoxia and ischemia. Real-Time Iontophoretic and Integrative Optical Imaging methods were used to evaluate diffusion transport in cortical slices under normoxic and ischemic conditions, and results were compared to values previously collected in rats. NM-R brains under normoxic conditions had a smaller ECS volume fraction than rats, and a correspondingly decreased diffusion permeability for macromolecules. Surprisingly, and in sharp contrast to rats, the NM-R ECS responded to ischemic conditions at the center of thick brain slices by expanding, rather than shrinking, and preserving diffusion permeabilities for small and large molecules. The NM-R thick slices also showed a blunted accumulation of ECS potassium compared to rats. The remarkable dynamic response of the NM-R ECS to ischemia likely demonstrates an adaptation for NM-R to maintain brain function in their extreme nest environment.

Introduction

The African Naked Mole-Rat (NM-R; Heterocephalus Glaber) is a eusocial fossorial rodent with activity centered around a densely populated colony nest. Because NM-R experience elevated carbon dioxide and reduced oxygen availability in their nest chambers (Zions et al., 2020), mitigating prolonged exposure to the diminished air quality is important for their survival. NM-R adaptations for conserving energy under hypoxic and hypercapnic conditions include systemic adaptations to reduce oxygen needs (Buffenstein and Yahav, 1991, Yahav and Buffenstein, 1991) and improve oxygen availability to tissues (Johansen et al., 1976). In the brain, adaptations (Larson and Park, 2009, Larson et al., 2014) include reduced neuronal ion transport (Zions et al., 2020), blunted neuronal and respiratory responses to hypercapnia (Park et al., 2008, Smith et al., 2011, Kirby et al., 2018, Clayson et al., 2020), and reduced dependence on oxidative metabolism (Park et al., 2017). Since extracellular space (ECS) is a compartment of the brain tissue vital for molecular transport, we asked whether the extracellular microenvironment of the NM-R brain adapts to low oxygen supply in order to sustain chemical intercellular signaling, nutrient delivery and metabolite removal in the adverse conditions of the nest environment.

Brain ECS provides the immediate external microenvironment of all brain cells, comprising roughly 20% of the total brain tissue volume (Nicholson and Hrabětová, 2017). It is filled with ionic fluid that closely resembles cerebrospinal fluid in composition and harbors extracellular matrix, a macromolecular assembly of glycans and proteins with complex structure and signaling properties (Dityatev et al., 2010, Smith et al., 2015). Brain ECS is important for brain function in four principal ways: 1) it forms a reservoir of ions that are indispensable for cell membrane polarization and neuronal activity, 2) it enables chemical signaling between brain cells by facilitating diffusion of small neurotransmitters and neuropeptides as well as larger proteins, 3) it provides a route for delivery of oxygen and glucose from the vasculature to the brain cells for their metabolic needs, and 4) it facilitates timely clearance of metabolites and harmful substances (Nicholson and Hrabětová, 2017). Brain ECS in the NM-R has not been systematically studied, although it has been reported that NM-Rs produce higher molecular weight variants of the extracellular matrix glycan hyaluronan (HMW-HA) (Tian et al., 2013, del Marmol et al., 2021) which form large supercoiled structures in the NM-R brain (Kulaberoglu et al., 2019). This enhanced HMW-HA scaffolding, coupled with comparatively low density of neurons and other cells in the adult NM-R brain (Herculano-Houzel et al., 2011), may play a role in adapting the extracellular microenvironment for enhanced delivery of oxygen and glucose to brain cells under extreme environmental conditions.

Structural properties of brain ECS can be deduced from biophysical experiments employing diffusion of biologically inert and membrane-impermeable molecular probes (Hrabĕtová and Nicholson, 2007). Analysis of such diffusion measurements primarily yields two structural parameters: 1) ECS volume fraction (α = V_ECS/V_total), which indicates the volume of total brain tissue occupied by ECS, and 2) diffusion permeability (θ = D*/D_free), which indicates the extent to which an effective diffusion coefficient D* of a particular molecular probe is reduced by obstacles and interactions in the extracellular microenvironment, compared to a diffusion coefficient D_free in a medium with no obstacles (Nicholson, 2001, Hrabe et al., 2004). Volume fraction has been measured across multiple species using Real-Time Iontophoresis (RTI) of a small cation tetramethylammonium (TMA⁺, MW 74, hydrodynamic diameter 0.5 nm) as a probe molecule (Nicholson and Phillips, 1981, Odackal et al., 2017). ECS volume fraction ranges from 15 to 30% across the lifespan, with a typical adult value of around 20% (Syková and Nicholson, 2008, Colbourn et al., 2019). The RTI method also obtains diffusion permeability for the same small TMA⁺ molecule. The permeability for larger molecules, such as the 3,000 MW dextran macromolecule (dex3, hydrodynamic diameter 2.1 nm), can be determined by Integrative Optical Imaging (IOI), a fluorescent microscopy technique which analyzes diffusion of fluorophore-tagged macromolecular probes through the brain ECS (Nicholson and Tao, 1993). In isotropic gray matter, a typical value of θ_TMA is about 0.4 while θ_dex3 is only about 0.3 (Nicholson and Phillips, 1981, Nicholson and Tao, 1993, Syková and Nicholson, 2008, Odackal et al., 2017), reflecting slower diffusional spread of macromolecules primarily due to the restricted diffusion effect (cellular wall drag) (Thorne and Nicholson, 2006, Hrabe and Hrabĕtová, 2019), and possibly increased interactions with the extracellular matrix (Godin et al., 2017, Soria et al., 2020).

Both ECS volume fraction and diffusion permeability are usually decreased under adverse conditions such as hypoxia and ischemia (Syková et al., 1994), as a result of water driven osmotically from the extracellular to the intracellular compartments of neurons and glia. In ischemia in vivo, rat ECS volume fraction reduces from 20% to as low as 5%, and diffusion permeability for TMA⁺ also significantly decreases (Syková et al., 1994, Voříšek and Syková, 1997). The effect of cellular swelling has been reproduced in vitro by the substitution of the extracellular fluid with a hypotonic solution (Hrabĕtová et al., 2003). Hypoxic conditions have been mimicked in a thick slice preparation (typically 1 mm) which restricts the supply of oxygen to the center of the slice (Fujii et al., 1982, Bingmann and Kolde, 1982, Newman et al., 1988, McGoron et al., 1997, Hrabĕtová et al., 2002). The thick slice preparation likely creates a pattern of global ischemia deep in the slice where tissue is required to undergo a metabolic shift with increased anaerobic metabolism (Newman et al., 1988). This metabolic shift, aside from the consequential lactic acid accumulation leading to extracellular acidification, also depletes ATP and results in production of free radicals by mitochondrial electron transport chain, intracellular Na⁺ accumulation, extracellular K⁺ accumulation, influx of excess Ca²⁺, and release of excitatory neurotransmitters such as glutamate (Newman et al., 1989, Newman et al., 1991, Newman et al., 1995, Hrabĕtová et al., 2002, Newman et al., 2002). Diffusion measurements in thick slice cortical samples from rats demonstrate a reduction of ECS volume fraction to approximately 10% and a significant decrease in diffusion permeability for both TMA⁺ and dex3 (Hrabĕtová et al., 2002, Hrabĕtová et al., 2003). Monitoring extracellular K⁺ documented a precipitous rise in K⁺ concentration when ECS volume fraction and diffusion permeability were reduced in ischemic conditions, indicating anoxic depolarization (Hrabĕtová et al., 2002).

In the present study, we investigated ECS in the cortex of adult NM-R in vitro, in normal and thick slice conditions. Compared to rats, our results show a diminished ECS volume and reduced diffusion permeability for the dex3 macromolecule in normal slices from NM-R brain. However, in thick slices, the ECS volume of NM-R does not collapse but significantly expands, in sharp contrast to ECS in the rat brain. This expansion results in a remarkable stability of diffusion permeability for both the dex3 macromolecule and the small TMA⁺ ion. These findings suggest that ECS adaptation represents a neuroprotective feature of NM-R brain to tolerate extreme environmental conditions.