Guanosine as a Potential Therapy on Traumatic Brain Injury

CDC defines TBI as a disruption in the normal function of the brain that can be caused by a bump, blow, or jolt to the head, or penetrating head injury. TBI is typically classified as focal (confined to a specific location), diffused (comprises a widespread area) or mixed injury (combination of both focal and diffused). Different aspects including TBI ethology, severity, brain region affected, and patient gender direct contribute to a unique brain pathology with a widely varying clinical outcome.

TBI pathophysiology is commonly classified into primary and secondary injury. Primary injury corresponds to the mechanical forces involved at impact moment inducing bone fractures, tissue and vascular damage as well as immediate cell death. As result, multiple pathophysiological mechanisms resulting in further brain damage are observed over hours or even years following the inciting TBI event, giving rise to term secondary injury(Ng & Lee, 2019). Secondary injury initially comprises multiple neurotoxic events including excitotoxicity, calcium influx, mitochondrial dysfunction, oxidative stress, neuronal death, inflammation and cerebral edema. Acute neurochemical changes observed in the first hours following TBI may be associated with the emergence of early behaviour impairments including motor deficit. On the other hand, long-term progression of secondary injury is followed by several changes underlying brain network reorganization. Thus, TBI patients may develop long-term neuropsychiatry complications including memory deficit and anxiety. In the next sections we are going to discuss the potential use of GUO on the acute/long-term progression of secondary injury and behaviour impairments following TBI.

Glutamate is considered the major excitatory neurotransmitter in the CNS playing an essential role on neuronal development, plasticity, memory and learning. Despite that, excessive levels of this amino acid in neuronal synapses may also result in neuronal death by excitotoxic mechanisms. Thereby, the precise balance of glutamate release and uptake are crucial to maintain the fine-tune of brain function.

A common TBI feature is the abrupt spreading depolarization of neurons at the damage epicentre resulting in a massive and disseminated glutamate release in the synaptic cleft (Katayama, Becker, Tamura, & Hovda, 1990). In addition, the glutamate clearance mainly performed by excitatory amino acid transporters GLAST and GLT1 in astrocytes are impaired during the initial stage following TBI (Dobrachinski et al., 2017). Preclinical studies show that a single GUO injection 15 or 40 min following FPI in rats, restore the glutamate uptake activity in hippocampal and cortical slices 3 and 8 hours upon TBI (Dobrachinski et al., 2017; R. D. R. Gerbatin et al., 2017). In addition, GUO prevents the FPI-induced downregulation on expression levels of glutamate transporters GLAST and GLT1 (Dobrachinski et al., 2017). Thus, GUO likely restore the glutamate uptake function by maintaining the suitable protein levels of glutamate transporters in astrocytes.

Glutamate uptake function is also tightly dependent of extracellular gradients of Na+ and cytoplasmic K+ sustained in equilibrium by the transmembrane enzyme Na+ K+-ATPase (Danbolt, 2001). GLT1 and GLAST drive the extracellular glutamate into astrocytes by a cotransport of high affinity of 3Na+ ions in the exchange of 1K+ ion (Danbolt, 2001). However, FPI results in loss of Na+ K+-ATPase activity leading to a breakdown of Na+ and K+ electrochemical gradients across membranes resulting in potential disruption of glutamate uptake function (R. D. R. Gerbatin et al., 2017). Interestingly, GUO protects against the loss on Na+ K+-ATPase activity in rats submitted to FPI (R. D. R. Gerbatin et al., 2017). This GUO effect likely reflects not only on maintenance of glutamate uptake function but also in cell osmotic equilibrium preventing astrocytic swelling induced by TBI.

Glutamate delivered into astrocytes is converted into glutamine by action of glutamine synthetase enzyme (GS) (Danbolt, 2001). Thereby, GS has an essential role for glutamate recycling sustaining this neurotransmitter below toxic levels in the CNS. FPI in rats results in failure of GS activity indicating a possible reduction of glutamate recycling in astrocytes exacerbating the harmful glutamate effects in the brain(R. D. R. Gerbatin et al., 2017). In contrast, GUO treated rats show a significant protection against the loss on GS activity 8 hours after FPI (R. D. R. Gerbatin et al., 2017). Thus, GUO seems to restore the glutamate recycling in astrocytes attenuating the glutamate toxicity in CNS.

Changes in the cellular redox state may also represents another important factor for the raise of glutamate levels observed following TBI. Oxidative damage targeting proteins such as GLT1, GLAST, Na+ K+-ATPase and GS may disrupt its function resulting in failure of precise synchrony between glutamate uptake and recycling in astrocytes (Hachimori et al., 1975; Morel, Tallineau, Pontcharraud, Piriou, & Huguet, 1998; Trotti, Danbolt, & Volterra, 1998). Interestingly, GUO shows protective effects against FPI-induced protein carbonyl content in rats (R. D. R. Gerbatin et al., 2017). The finding suggests GUO might also control the glutamate homeostasis in CNS by attenuating the TBI-induced oxidative damage to proteins involved in glutamate uptake/recycling in astrocytes.

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Increased levels of glutamate in the synaptic cleft following TBI are associated with a massive Ca2+ and Na+ influx into neuronal and glial cells resulting from an overstimulation of NMDA and AMPA receptors (Ng & Lee, 2019). Cytoplasmic Ca2+ homeostasis in these cells is constantly maintained by mitochondria through Ca2+ sequester into the mitochondrial matrix compartment (Ng & Lee, 2019). However, Ca2+ overload into this compartment may results in mitochondrial swelling. Mitochondrial swelling following FPI in rats has been associated with loss of mitochondrial potential (ΔΨm), ROS generation and unbalance of redox system (Dobrachinski et al., 2017). In a closed-head model of mild TBI in rats, mitochondrial bioenergetic function evaluated by high-resolution respirometry (HRR) was also found compromised after TBI (Courtes et al., 2020). In both TBI models, all parameters related to mitochondrial respiration were restored by a single GUO treatment following TBI (Courtes et al., 2020; Dobrachinski et al., 2017; R. D. R. Gerbatin et al., 2017). In addition, such GUO effect on mitochondrial function was associated with the modulation of A1 adenosine receptor (R. R. Gerbatin, Dobrachinski, Cassol, Soares, & Royes, 2019). Thus, GUO likely sustain the suitable balance of mitochondrial function across different TBI models by reducing mitochondrial calcium overload in a dependent manner of A1 adenosine receptor modulation.

Mitochondrial disfunction is also associated with cell death by necrotic (lack of ATP) or apoptotic events following TBI (Ng & Lee, 2019). The opening of the mitochondrial permeability transition pore (MPTP) and release of cytochrome C may lead to caspase 3 activation (Ng & Lee, 2019). As a main effector apoptotic protein, caspase 3 initiates the programmed cell death from mitochondrial (intrinsic pathway) or cytoplasmic signalling (extrinsic pathway) (Ng & Lee, 2019). In contrast, GUO treatment after FPI attenuates the spread of neuronal loss around the injury site and protects against an increase in the levels of caspase 3 (R. D. R. Gerbatin et al., 2017). The finding suggests GUO may effectively counteract the progress of neurotoxic events associated to TBI.

TBI-induced breakdown of blood brain barrier and cellular damage results in an immediate neuroinflammatory response characterized by several inflammatory mediators including TNFα and IL- β (Ng & Lee, 2019). Exacerbated levels of both pro-inflammatory mediators is critically related with apoptotic events and formation of cerebral edema resulting in extension of neuronal loss (R. D. R. Gerbatin et al., 2017; Ng & Lee, 2019). GUO treatment 40 min following FPI seems to reduce the levels of TNFα and IL-1 beta 8 hours after TBI in rats. This GUO effect was associated with reduction of edema in the perilesional area of TBI what potentially contributes to the preservation of neurological functions.

A typical behaviour impairment observed immediately following TBI comprehend the motor deficit. Motor function is mediated from cortex to spinal cord reaching the skeletal muscle through a precise signalling involving several brain regions including cortex, sensorimotor cortex, subcortical nuclei, cerebellum and brainstem (Fujimoto et al., 2004). Thereby, lacerations or typical neurochemical changes previously mentioned in any of these brain regions may disrupt the complex signalling to coordinate the movement. FPI in rats results in an early reduction of spontaneous locomotion activity characterized by a decrease in the number of crossing, rearing, velocity and distance travelled in the open field. On the other hand, GUO treatment upon FPI restores all parameters related to spontaneous locomotion to control levels. Furthermore, the motor coordination deficit observed following the mild TBI (closed-head model) and FPI in rats, was also prevented with a single GUO treatment. Taken together, GUO neuroprotective effects against early TBI-induced motor deficit may reflect the ability of GUO to blunt multiple neurotoxic pathways underlying the secondary injury following TBI.

The progression of secondary injury into a chronic phase may result in several neuropsychiatry complications including memory deficit and anxiety. Rats submitted to FPI show anxiety-like behaviour and memory deficits at 14 and 21 days following TBI, respectively (Dobrachinski et al., 2019). In contrast, GUO daily treatment after FPI prevents the development of anxiety-like behaviour traits and loss of memory performance (Dobrachinski et al., 2019).

Such long-term behaviour impairments following TBI may result from molecular and morphological changes underlying synapse network reorganization in different brain regions. In fact, changes in CREB-BDNF signalling in the hippocampus may compromise the synaptic plasticity of hippocampal neurons resulting in cognitive and emotional disorders. FPI in rats induces a long-term downregulation of BDNF and CREB in hippocampal neurons 21 days after TBI (Dobrachinski et al., 2019). Chronic GUO treatment over the same period prevents such decrease of both genes (Dobrachinski et al., 2019). In addition, GUO blunts the FPI-induced decrease of a calcium-binding protein found in the membrane of synaptic vesicles in hippocampus (synaptophysin). However, GUO did not show any effect on the expression levels of GAP-43, a protein related with synapse repair (Dobrachinski et al., 2019). Therefore, these data suggest that GUO prevents emotional problems and memory deficits following TBI by attenuating synaptotoxicity in hippocampal neurons.

From a morphological perspective, synapse function may be also negatively affected by reactive gliosis triggered by TBI (Sajja, Hlavac, & VandeVord, 2016). Gliosis is characterized by long-term morphological changes in astrocytes and microglia, which is thought to also contribute for mood swings and cognitive decline after TBI (Sajja et al., 2016). Accordingly, FPI in rats induces a reactive astro- and micro-glioses in the hippocampus while chronic GUO treatment counteracted these morphological changes (Dobrachinski et al., 2019). Taken together, GUO shows an ability in preventing molecular and morphological changes in hippocampus associated to emotional problems and memory deficits following TBI. Interestingly, GUO neuroprotective effects in TBI seems to be dependent on the modulation of A1 adenosine receptors (Dobrachinski et al., 2019). In the next section we are going to discuss the involving of purinergic signalling on the neuroprotective effects of GUO.