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Enhanced nociceptive behavior and expansion of associated primary afferents in a rabbit model of cerebral palsy.

ABSTRACT: Spastic cerebral palsy (CP) is a movement disorder marked by hypertonia and hyperreflexia; the most prevalent comorbidity is pain. Since spinal nociceptive afferents contribute to both the sensation of painful stimuli as well as reflex circuits involved in movement, we investigated the relationship between prenatal hypoxia-ischemia (HI) injury which can cause CP, and possible changes in spinal nociceptive circuitry. To do this, we examined nociceptive afferents and mechanical and thermal sensitivity of New Zealand White rabbit kits after prenatal HI or a sham surgical procedure. As described previously, a range of motor deficits similar to spastic CP was observed in kits born naturally after HI (40 min at ~70%-80% gestation). We found that HI caused an expansion of peptidergic afferents (marked by expression of calcitonin gene-related peptide) in both the superficial and deep dorsal horn at postnatal day (P)5. Non-peptidergic nociceptive afferent arborization (labeled by isolectin B4) was unaltered in HI kits, but overlap of the two populations (peptidergic and non-peptidergic nociceptors) was increased by HI. Density of glial fibrillary acidic protein was unchanged within spinal cord white matter regions important in nociceptive transmission at P5. We found that mechanical and thermal nociception was enhanced in HI kits even in the absence of motor deficits. These findings suggest that prenatal HI injury impacts spinal sensory pathways in addition to the more well-established disruptions to descending motor circuits. In conclusion, changes to spinal nociceptive circuitry could disrupt spinal reflexes and contribute to pain experienced by individuals with CP.

SUBMITTER: Reedich EJ

PROVIDER: S-EPMC9388620 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Enhanced nociceptive behavior and expansion of associated primary afferents in a rabbit model of cerebral palsy.

Reedich Emily J EJ Genry Landon T LT Singer Meredith A MA Cavarsan Clarissa Fantin CF Mena Avila Elvia E Boudreau Daphne M DM Brennan Michael C MC Garrett Alyssa M AM Dowaliby Lisa L Detloff Megan R MR Quinlan Katharina A KA

Journal of neuroscience research 20220715 10

Spastic cerebral palsy (CP) is a movement disorder marked by hypertonia and hyperreflexia; the most prevalent comorbidity is pain. Since spinal nociceptive afferents contribute to both the sensation of painful stimuli as well as reflex circuits involved in movement, we investigated the relationship between prenatal hypoxia-ischemia (HI) injury which can cause CP, and possible changes in spinal nociceptive circuitry. To do this, we examined nociceptive afferents and mechanical and thermal sensiti ...[more]

PMID: 35839339

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Project description:Cerebral palsy (CP) is caused by a variety of factors that damage the developing central nervous system. Impaired motor control, including muscle stiffness and spasticity, is the hallmark of spastic CP. Rabbits that experience hypoxic-ischaemic (HI) injury in utero (at 70%-83% gestation) are born with muscle stiffness, hyperreflexia and, as recently discovered, increased 5-HT in the spinal cord. To determine whether serotonergic modulation of spinal motoneurons (MNs) contributes to motor deficits, we performed ex vivo whole cell patch clamp in neonatal rabbit spinal cord slices at postnatal day (P) 0-5. HI MNs responded to the application of α-methyl 5-HT (a 5-HT1 /5-HT2 receptor agonist) and citalopram (a selective 5-HT reuptake inhibitor) with increased amplitude and hyperpolarization of persistent inward currents and hyperpolarized threshold voltage for action potentials, whereas control MNs did not exhibit any of these responses. Although 5-HT similarly modulated MN properties of HI motor-unaffected and motor-affected kits, it affected sag/hyperpolarization-activated cation current (Ih ) and spike frequency adaptation only in HI motor-affected MNs. To further explore the differential sensitivity of MNs to 5-HT, we performed immunostaining for inhibitory 5-HT1A receptors in lumbar spinal MNs at P5. Fewer HI MNs expressed the 5-HT1A receptor compared to age-matched control MNs. This suggests that HI MNs may lack a normal mechanism of central fatigue, mediated by 5-HT1A receptors. Altered expression of other 5-HT receptors (including 5-HT2 ) likely also contributes to the robust increase in HI MN excitability. In summary, by directly exciting MNs, the increased concentration of spinal 5-HT in HI-affected rabbits can cause MN hyperexcitability, muscle stiffness and spasticity characteristic of CP. Therapeutic strategies that target serotonergic neuromodulation may be beneficial to individuals with CP. KEY POINTS: We used whole cell patch clamp electrophysiology to test the responsivity of spinal motoneurons (MNs) from neonatal control and hypoxia-ischaemia (HI) rabbits to 5-HT, which is elevated in the spinal cord after prenatal HI injury. HI rabbit MNs showed a more robust excitatory response to 5-HT than control rabbit MNs, including hyperpolarization of the persistent inward current and threshold voltage for action potentials. Although most MN properties of HI motor-unaffected and motor-affected kits responded similarly to 5-HT, 5-HT caused larger sag/hyperpolarization-activated cation current (Ih ) and altered repetitive firing patterns only in HI motor-affected MNs. Immunostaining revealed that fewer lumbar MNs expressed inhibitory 5-HT1A receptors in HI rabbits compared to controls, which could account for the more robust excitatory response of HI MNs to 5-HT. These results suggest that elevated 5-HT after prenatal HI injury could trigger a cascade of events that lead to muscle stiffness and altered motor unit development.

Project description:BackgroundNeuroinflammation mediated by microglia plays a central role in the pathogenesis of perinatal/neonatal brain injury, including cerebral palsy (CP). Therapeutics mitigating neuroinflammation potentially provide an effective strategy to slow the disease progression and rescue normal brain development. Building on our prior results which showed that a generation-4 hydroxyl poly(amidoamine) (PAMAM) dendrimer could deliver drugs specifically to activated glia from systemic circulation, we evaluated the sustained efficacy of a generation-6 (G6) hydroxyl-terminated PAMAM dendrimer that showed a longer blood circulation time and increased brain accumulation. N-acetyl-L-cysteine (NAC), an antioxidant and anti-inflammatory agent that has high plasma protein binding properties and poor brain penetration, was conjugated to G6-PAMAM dendrimer-NAC (G6D-NAC). The efficacy of microglia-targeted G6D-NAC conjugate was evaluated in a clinically relevant rabbit model of CP, with a mild/moderate CP phenotype to provide a longer survival of untreated CP kits, enabling the assessment of sustained efficacy over 15 days of life.MethodsG6D-NAC was conjugated and characterized. Cytotoxicity and anti-inflammatory assays were performed in BV-2 microglial cells. The efficacy of G6D-NAC was evaluated in a rabbit model of CP. CP kits were randomly divided into 5 groups on postnatal day 1 (PND1) and received an intravenous injection of a single dose of PBS, or G6D-NAC (2 or 5 mg/kg), or NAC (2 or 5 mg/kg). Neurobehavioral tests, microglia morphology, and neuroinflammation were evaluated at postnatal day 5 (PND5) and day 15 (PND15).ResultsA single dose of systemic 'long circulating' G6D-NAC showed a significant penetration across the impaired blood-brain-barrier (BBB), delivered NAC specifically to activated microglia, and significantly reduced microglia-mediated neuroinflammation in both the cortex and cerebellum white matter areas. Moreover, G6D-NAC treatment significantly improved neonatal rabbit survival rate and rescued motor function to nearly healthy control levels at least up to 15 days after birth (PND15), while CP kits treated with free NAC died before PND9.ConclusionsTargeted delivery of therapeutics to activated microglia in neonatal brain injury can ameliorate pro-inflammatory microglial responses to injury, promote survival rate, and improve neurological outcomes that can be sustained for a long period. Appropriate manipulation of activated microglia enabled by G6D-NAC can impact the injury significantly beyond inflammation.

Dataset Information

Enhanced nociceptive behavior and expansion of associated primary afferents in a rabbit model of cerebral palsy.

Publications

Enhanced nociceptive behavior and expansion of associated primary afferents in a rabbit model of cerebral palsy.

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