Overall, the findings from this study demonstrated that female monkeys evidenced less severe impairment of fine motor function of the hand and digits after a cortical injury in primary motor cortex than male monkeys even though the volume of the lesions did not differ. Specifically, female monkeys showed less severe impairments early in the recovery period and a more complete recovery of grasp function than male monkeys.
While a return to pre-injury levels of motor performance is rare in animal models and humans, it was of particular interest in the present study that four of the five female monkeys did return to a precise finger-thumb grasp rather in place of the compensatory grasp patterns observed in the male monkeys. While the development of a compensatory grasp pattern by the male monkeys did achieve the goal of retrieving the food reward on our motor task, this type of compensatory grasp, which is often observed in human stroke patients, is inefficient and does not translate into effective fine motor function of the digits that is required for successful completion of activities of daily living. Therefore, determining the underlying mechanisms of the less severe impairment and the greater degree of recovery of grasp pattern in the female monkeys is of critical importance.
Sex differences in the pathogenesis of cortical injury
The pathogenesis of cortical injury is characterized by a cascade initiated by acute cellular damage, eventually leading to a sustained inflammatory response, chronic accumulation of oxidative stress, and secondary damage, which impairs cortical reorganization and recovery [49–52]. The brain inflammatory cells, microglia and astrocytes, release several cytotoxic agents including matrix metalloproteinases, nitric oxide, and reactive oxygen species (ROS) which lead to cell death [51,53–55]. The immune response following cortical injury involves increases in reactive astrocytes and microglia that produce ROS and inflammatory cytokines and chemokines [50–52,54,56], which can also disrupt neuronal recovery and reorganization.
A large body of evidence from studies in rodents demonstrates sex differences in these immune responses to cortical injury [12,22,24–26,57]. In rodents, a diminished pro-inflammatory response associated with a lower density of amoeboid (activated) microglia was found in females compared to males after recovery from ischemic injury [58–60]. Additionally, male neurons and astrocytes are more sensitive to ischemia and oxygen-glucose deprivation than female cells in vitro [57,61–63]. In response to the inflammatory agent, lipopolysaccharide, male astrocytes show enhanced expression of pro-inflammatory cytokines, IL6, TNF-alpha and IL1B, while female astrocytes show enhanced expression of the anti-inflammatory cytokine, IL10 [64,65]. Further, there is decreased ROS production in astrocytes in female brains compared to male brains [66–68]. In addition to these inflammatory responses, studies with cell cultures have demonstrated sex specific neuronal death mechanisms. For instance, cell death after ischemia in primary neuronal cultures derived from male brains are mediated by excessive ROS production and over-activation of poly(ADP)ribose polymerase (PARP) while death of cells from female brains involves programmed caspase-dependent apoptosis [27,69–73].
One potential mechanism underlying age-dependent sex differences is the release of estrogen following brain injury. As a neuroprotective agent, E2 acts on ER-a, and ER-b receptors expressed by neurons, astrocytes, and microglia to modulate inflammation and promote plasticity [12,16,22,24,25,74]. Acute estradiol treatment increases spine density and stabilization of newly formed spines through recruitment of synaptic proteins and receptors in cultured cortical neurons . In rodent and monkey studies, estradiol treatment has shown to play an important role in rescuing age-related synaptic plasticity by modulating actin and synapse formation [75–77]. In addition, estrogen stimulates neuronal survival by altering the expression of the anti-apoptotic gene, bcl-2 that inhibits free radical formation . Further, estrogen can reduce inflammation through interactions with neurotrophic factors and by directly acting on the ER-a receptors on astrocytes and microglia [12,24,26,66,78–80]. Astrocytes express estrogen receptors and produce estradiol in both males and females [80,81]. Following cortical injury, astrocyte-derived estradiol mediates anti-inflammatory effects through the release of neurotrophic factors, BDNF, IGF-1, and GLT-1, as the astrocytes become reactive and increase expression of ER-a receptors and glial fibrillary acidic protein (GFAP) [9,23,74,78,82–86].
Estrogen levels in aged rhesus monkeys
While there is substantial evidence that acute estrogen treatment in vitro and increased estrogen after injury in vivo in rodents lead to neuroprotection, how the cyclical changes in estrogen influence susceptibility to injury is not clear. Here we show no significant linear relationships between pre-operative, baseline estrogen levels at the time of cortical injury and any of the recovery measures, which suggests that undetected multivariate cyclical hormonal factors may be a factor. There are varied definitions of menopause in humans, ranging from criterion such a permanent cessation of menstruation and cessation of steroid hormone secretion [87,88]. However, what is most commonly considered criterion for menopause is the permanent, non-pathologic, age-associated cessation of ovulation as measured by increases in follicle stimulating hormone (FSH) couple with decreases in anti-Mullerian hormone (AMH), inhibin B, and estradiol [89,90]. Using this criterion, most studies place the mean age of menopause in humans at approximately 50 years of age. However, the criterion and age of onset of menopause and age-associated hormonal levels are less clear in the rhesus monkey due to the effects of captivity, controlled laboratory and housing environments, and seasonal breeding in this species . Given these challenges, estimates for age of menopause in captive rhesus monkeys have ranged from 22-27 years of age [91–93]. The monkeys in the current study ranged from 20-26 years of age and were therefore likely undergoing age-related reductions in FSH, AMH, inhibin B, and estradiol [87,93–95] which may in part explain why there was a no significant linear relationship between degree of recovery and estrogen levels at time of cortical injury. Further, we did not monitor post-operative estrogen levels throughout the recovery period and therefore cannot assess whether increases in estrogen levels induced by injury may have facilitated recovery.
Sex Differences in Human Stroke and Recovery
While our model does not replicate stroke per se, it does model the injury and inflammatory cascade that occurs following stroke and therefore provides insight about recovery of function, plasticity, and repair in the brain and potential sex differences in these recovery processes. Regarding sex differences in humans, there is a higher incidence of stroke in males until the age of 65 years. Afterwards, the prevalence and severity of stroke among females significantly increases [2,4–6,8,96]. This shift during advanced age is attributed to decreases in estrogen and its corresponding neuroprotective effects, especially 17ß-estradiol (E2) .
Clinical studies demonstrate considerable evidence that post-menopausal females experience greater stroke severity than their male counterparts, but differences in functional recovery remain unclear. For example, it has been shown that males experience a greater relative loss of muscle strength than females when post-stroke upper extremity muscle strength in the affected limb is reported as a percentage of strength of the unaffected side . However, most studies demonstrate an overall lesser degree of physical recovery in females [2,4,96,98–101]. The degree of recovery after stroke or other brain injuries is often assessed in large clinical trials by evaluating activities of daily living (ADL) using the Barthel Index (BI) or the modified Rankin scale (mRS) . Both scales are widely used, but are frequently challenged by questions of subjectivity, reliability, and sensitivity [102,103]. In addition, the scales do not assess the level of return to pre-injury motor function, but instead only measure independence level while performing daily tasks or routines. Since males typically experience stroke at a younger age than females, the difference in scores is often explained by males having more assistance with ADLs from a spouse, while females are more likely to be widowed at the time they experience a stroke [2,100,104]. Thus, the independence levels reported by women are often lower and associated with less assistance at home to perform their tasks . Clinical studies are further challenged by other social and biological factors that include confounding factors such as mental health, lifestyle, comorbid risk factors (i.e., cardiovascular disease, diabetes mellitus, hypertension, etc.), household expectations, and family support [4,6].
Perspectives and Significance
Currently, there is no consensus on outcome measures that can provide a more complete and quantitative assessment of post-stroke recovery in human females compared to males . Despite the clinical and epidemiological evidence of sex differences, the variability and confounding factors inherent to clinical studies affirms the importance of translatable models that can provide quantitative analyses of functional impairment and recovery after injury.