Resumen de The central noradrenergic system in a kaolin-induced model of hydrocephalus: Implications for pain modulation and micturition control
Hydrocephalus is a neurological disease characterized by the disturbed circulation of cerebrospinal fluid (CSF), which results in dilation of the cerebral ventricular system, with an impact on the nervous tissue that surrounds the ventricles. Some of the central structures involved in pain modulation and neuronal control of micturition have a periventricular location, namely the periaqueductal grey (PAG) and the locus coeruleus (LC).
Pain transmission at the spinal cord is modulated by descending projections that arise from supraspinal areas which collectively form the endogenous pain control system. The PAG has an essential role in descending pain modulation, receiving information from higher centers in the brain and sending outputs to the spinal cord, through relay stations, including the LC. Concerning the LC, it is involved in descending pain modulation through direct noradrenergic projections to the spinal cord.
The functions of the lower urinary tract (LUT) are dependent on neuronal circuits located in the brain and spinal cord, being organized in a switch-like pattern of activity that turns on and off, in an all-ornone way. Sensitive information from the bladder reaches the PAG through spinal projections, using the pontine micturition center (PMC) as a relay station. Projections from the PMC diverge to innervate the LC, reaching the neurons of the Onuf's nucleus, allowing the synchronization of contraction and relaxation of the bladder and external urethral sphincter (EUS), respectively, during voiding. Regarding the neural control of micturition, the normal micturition reflex is dependent on the spinobulbospinal reflex that involves the PAG, the PMC, and the spinal cord, which is regulated by higher brain structures such as the frontal cortex and the basal ganglia. An interaction between the PAG and PMC results in efferent signals to the spinal cord which generates the appropriate motor responses necessary for 13 voiding.
It remains to be evaluated if the hydrocephalus-induced damage to periventricular brain areas (PAG and LC) may account for impairments in descending pain modulation and neuronal control of LUT. In the two studies included in this thesis, we investigated noradrenergic pathways during hydrocephalus, focusing on the implications in descending pain modulation and in the neuronal control of micturition.
To answer these questions, we used a validated hydrocephalus model the rat injected with kaolin into the cisterna magna. Collaborations with neurosurgeons from the Neurosurgery Service of Hospital de S. João (Porto, Portugal) and the Department of Neurosciences and Mental Health of the Faculty of Medicine of Porto were established for the first study. For the second study, we collaborated with experts in neuro-urology at the Institute of Research and Innovation in Health of the University of Porto, Portugal.
In the publication I (International Journal of Molecular Sciences; Quartile 1) we evaluate the effects of kaolin injection; we measured the degree of ventricular dilatation in sections encompassing the PAG by standard cytoarchitectonic stains (thionine staining); in general, rats with kaolin-induced hydrocephalus presented a higher dilatation of the 4th ventricle, along with a higher area of the PAG.
To evaluate the effects of hydrocephalus in pain modulation, four weeks after hydrocephalus induction, animals were sacrificed and immunodetection of the noradrenaline-synthetizing enzymes tyrosine hydroxylase (TH) was performed, at the LC and spinal cord, and dopamine beta-hydroxylase (DBH) in spinal cord. Hydrocephalic rats presented increases in the levels of TH in the LC; concerning the spinal cord, both TH and DBH levels were increased. The expression of a validated oxidative marker (8- Hydroxyguanosine; 8-OHdG) was studied in noradrenergic LC neurons by double immunodetection of TH and 8-OHdG. Hydrocephalic animals presented increases of 8-OHdG in the population of TH- 14 immunoreactive neurons of the LC. The following pain-related parameters were measured, namely 1) pain behavioral responses in a validated pain inflammatory test (the formalin test) and 2) the nociceptive activation of spinal cord neurons. A decrease in behavioral responses was detected in rats with kaolin-induced hydrocephalus, namely in the second phase of the test (inflammatory phase). A decrease in the number of neurons immunoreactive for Fos, a marker of neuronal activation, was detected in rats injected with kaolin, namely at the ventrolateral PAG (vlPAG), whereas the remaining PAG columns did not present statistically significant changes in the numbers of Fos-immunoreactive neurons.
The results of the behavioral studies indicate that rats with kaolin-induced hydrocephalus exhibit hypoalgesia. A decrease in Fos expression was detected at the superficial dorsal layers of the spinal cord in rats with kaolin-induced hydrocephalus, further indicating that hydrocephalus is associated with decreased nociceptive responses. This initial study indicates that pain control may be altered during hydrocephalus due to changes in the noradrenergic LC, involving oxidative stress events, with a consequence in descending pain modulatory circuits.
In publication II (submitted) we focused our studies on the control of micturition in hydrocephalic rats.
We evaluated this issue since urinary incontinence is frequently reported during hydrocephalus in patients, affecting the quality of life and being a significant bother for both patients and their caregivers.
We used the kaolin-induced hydrocephalus rat model referred above to study the mechanisms involved in the urinary dysfunction during hydrocephalus, focusing on two circumventricular areas: the LC and the PAG. Based on the abovementioned neural control of micturition, we started by studying neuronal activation of the PAG in hydrocephalic animals. A decrease in the number of Fos-immunoreactive cells in the group of hydrocephalic animals was detected, namely in the vlPAG column. The expression of 15 DBH in spinal cord sections from the L6 level was evaluated by immunohistochemistry. Hydrocephalic animals showed higher numbers of fibers immunoreactive to DBH. Eight weeks after hydrocephalus induction, the bladder function was evaluated by cystometries. Hydrocephalic animals showed an increase both in the number of bladder contractions and the minimum pressure to void. These results suggest alterations in the brain-bladder control network leading to an exaggerated micturition reflex, which may be due to impairments in noradrenergic descending pathways, possibly due to a decrease in the control of the vlPAG over the LC. The increased availability of noradrenaline production from the LC may account for the increased levels of noradrenaline at the spinal cord, namely in the Onufs nucleus causing an exaggeration of the micturition reflex leading to urinary impairments during hydrocephalus.
Collectively, the 2 studies included in this thesis indicate major changes in the brain tissue during hydrocephalus, with an impact on the PAG and LC. We propose that due to the impairment in CSF pathways and subsequent ventricle dilation, some brain areas may be hypoxic leading to an oxidative stress imbalance, and ultimately to a state of oxidative stress, which was demonstrated in the LC of hydrocephalic rats. Oxidative stress may induce increases in TH levels in the LC in an attempt to protect the neurons from oxidative thereat. It would be important to evaluate the occurrence of oxidative stress in other brain areas, namely the PAG, which also shows major alterations during hydrocephalus, namely structural and functional. The consequences of hydrocephalus in pain modulation and micturition control detected in kaolin-induced hydrocephalus rats need to be further studied, namely in what concerns antioxidative protective strategies to prevent the deleterious effects of the disease.
