Neurodevelopment: In this work we assessed physical and neurodevelopmental parameters during the early life period of the SHR strain confirming previous observations from other groups describing alterations in the maturation of neurological reflexes and motor coordination deficits. Bassan et al  stated that the model of pregnant SHR closely simulate human IUGR caused by hypertension in pregnancy and should enable investigation of mechanisms of hypertension-mediated placenta-vascular injury. The SHR pups exhibited significant neurodevelopmental differences in the acquisition of neonatal reflexes (righting, negative geotaxis, gait) accomplishing the tasks at earlier postnatal age comparing to the pups of the control strain but, on the contrary present a delayed onset of puberty, also consistent with previous studies demonstrating that ovarian development, steroidogenesis, and responsiveness to gonadotropins are strongly compromised in these animals . The precocious onset and restless responses observed in the righting reflexes, gait, negative geotaxis and the increases in foot faults observed in the SHR pups throughout the first weeks of life, may be linked to failure in the proper establishment and consolidation of the neuronal pathways involved in motor control. From their anxious phenotype, SHR strain has also been considered as an animal model of ADHD, since they ‘naturally’ display the main ADHD symptomatology such as hyperactivity and impulsivity , and in concordance, poor foetal growth increased the risk of ADHD in children [27; 28].
Increased foetal and neonatal mortality and morbidity as well as adult pathologic conditions are often associated to placental insufficiency [29; 30]. Pregnant Stroke Prone SHR displayed a significant reduction of placental mass and signs of placental insufficiency (i.e. hypertrophy and reduced branching morphogenesis of the labyrinth layer), associated with decreased offspring weights and increased cephalization index, which was also a common feature of the IUGR infants . This response is associated to the physiological adaptation to preserve oxygen and nutrient supply to the brain (brain-sparing phenomena) . In the human IUGR foetuses due to placental insufficiency, the cerebellar to supratentorial volume ratio, calculated by MRI images, is affected . In several species, rodents, guinea pig and sheep IUGR subjects are characterized by poor neuro-development and since the earlier studies of Altman  the experimental neurogenetic studies in the rat have established that all the cerebellar granule cells and a very high proportion of hippocampal granule cells are produced during the early postnatal period. The risk for behavioural disorders may be highest at late stages of foetal development, in prematurely born and small-for-age infants. Cerebellar lobule morphogenesis depends on conserved genetic mechanism to achieve the correct number of folds and also on local environmental clues, as for example, the Shh signal coming from the Purkinje cells (PC). Thus, while several malformations have a genetic basis, inflammation, fetal hemorrhage, and prematurity are often contributing factors . In this study we did not include the assessment of the PC’s because we did not observe anomalies in these cells using the routine laboratory stains. PC´s anomalies were described in a previous work of our laboratory on P15 Wistar rat pups after acute postnatal hypoxia-ischemia. Disrupted BG processes labelled with vimentin, another cytoskeleton marker, were observed in the hypoxic-rat pups at P15 , but alternatively now we describe the apparent fragmentation of the BG processes when labelled with the GFAP glial marker. Sampson et al. , reported decreased foetal brain levels of glial fibrillary acidic protein (GFAP) and α-internexin during gestation in thyroidectomised animals, another condition affecting foetal growth and related to glial cell maturation. The apparent deficits in the intermediate filament scaffold of the SHR could be in correspondence to the delayed cerebellar foliation and a probable defect in the granular neurons migration from the EGL towards the IGL via the glial guidance inferred by the differences in the thickness of the cerebella cortical layers during postnatal development . In addition, other rat models of IUGR also showed Bergmann glia disorganization at P7 and by P35, with a 10% decrease in Bergmann glial fibre density . We did not observe a decrease in the number of BG fibres and the granular neurones migration was not documented in this work. The observed fragmentation of the GB processed could be interpreted as a delayed exchange of the immature vimentin containing filaments to the more developed GFAP containing cellular processes, however, this issue still remains to be investigated. Recently it was described that Yap/Taz, two well-established downstream effectors of the Hippo pathway, known to regulate organ size by directing proliferation and apoptosis, and highly expressed in radial glia are playing a crucial role in establishing the radial scaffold and cellular polarity of neural progenitors during embryogenesis. Other effectors such as En2 , Jagged 1 , Atoh1 , Pard3a/JamC  have also been characterized among others, in mutant mice and preterm pigs delivered by C-section, presenting cerebellar layering alterations. In further work it would be worth to explore the status of these effectors in the developing SHR in an attempt to explain the images shown here.
IGF-1: Insulin-like peptides in the brain participate in neurogenesis. The expression of IGF-1 is associated with regeneration and angiogenesis both in normal development and in pathological cases such as hypoxia. In the human a decrease of IGF-1 function, causes severe intrauterine growth restriction as well as microcephaly, sensorineural deafness, developmental delay, and metabolic abnormalities . Under normal conditions the IGF-1 E2c variant predominates in cells differentiated into neurons. It expression decreases progressively in the first two postnatal weeks and increases under hypoxic conditions . The parenchymal expression of IGF-1 and its splice variants can be particularly useful to assess the status of the SHR newborns. In the cerebellum of the SHR we observed a sustained low expression of the mRNA IGF-1Ec variant during the first and second postnatal weeks, in contrast to the WKY, which presented a small but significantly higher expression at P7. The delayed organ development and altered neurological parameters observed in the SHR together with a lower expression of this neurotrophic factor could be interpreted as a probable lower amount of differentiated neurons than the control strain, in the cerebellum at equivalent postnatal ages.
Brain microvasculature: Studies on the SHR microvasculature were mostly reported on the aging SHR brain . Adult animals with established hypertension and “young animals”, going as far back as 6-7 months old SHR and WKY are reported in the literature . Previous morphometric analysis of capillary volume fraction, number of branches, capillary diameter and total length in adult SHRs with established hypertension concluded that those parameters were not significantly different among strains [47, 48]. We applied the Masson trichrome stain to our samples of brain tissues from WKY and SHR at P14. This staining technique is usually used for assessing the presence of collagen fibres and fibrin. No signs of fibrosis or anomalous collagen deposits were observed in our very young SHR, before the initiation of hypertension. However, the stained sections for optic microscopy from both, white and grey matter and from the pial vasculature of SHR, revealed the presence of blood vessels, with diverse diameters, some of them remarkably dilated. Increased capillary diameter together with hyperplasia, abnormal shape and ultrastructure, changed cellular distribution of junctional proteins in the endothelial cells together with a decreased number of pericytes are considered as signs of transendothelial permeability, as it was described in PDGF-B/PDGFR-b knock out mice .
In an attempt to deepen the comparative analysis among strains of the brain capillaries we applied two recognized endothelial cells markers, CAV-1 and Isolectin B4 to the same tissue slices. CAV-1 is a membrane integral protein located to caveolae believed to play an important role in the regulation of multiple cellular processes, including cell growth, differentiation, endocytosis, cholesterol trafficking, and cellular senescence . CAV-1 is thought to interact with occludin preventing the degradation of the structural proteins of the tight junctions (TJ) in the endothelial cells [51; 52]. On the other side, CAV-1 increases the BBB permeability via caveolae-based transcytosis and translocation of TJ protein. CAV-1 also inhibits matrix metalloproteinase-9 (MMP-9) which disrupts TJ proteins and basement membrane which means that CAV-1 may protect BBB integrity under certain circumstances, such as ischemic stroke. CAV-1 is also essential for NO-mediated and VEGF-induced angiogenesis, and there are evidences that Cav-1/VEGF-dependent pathway is important for neurogenesis, dendritic growth and arborisation .
In the human cerebral cortex CAV-1 is expressed by all the cell types that form the BBB, endothelial cells, pericytes, and vascular astrocytes . A decrease in the expression of CAV-1 in the brain capillaries at early postnatal life, would imply a disruption of the mechanisms mentioned above, affecting the normal function of the NVU.
Bandeiraea simplicifolia as other lectins binds with high affinity to α-galactosyl residues of glycoproteins on the cell surface of the endothelium thereby allowing the visualization of blood vessel endothelial cells including endothelial phalanx, stalk and tip cells, as well as endothelial tip cell filopodia . We analysed the expression of these two markers independently and in the combined form, and in this way we were able to observe differences among WKY and SHR in several parameters that characterize the brain cortex and cerebellar microvessels. These differences in general show a reduced amount of CAV-1 protein expression in the SHR’s brain microvessels. On the contrary, IB4 labelling seems to run in parallel during the two first postnatal weeks, indicating the presence of an active process of angiogenesis sprouting and branching of the newly formed vessels in the process of forming the mature CNS vessel network in both rat strains.
AngioTool is commonly applied to assess the superficial plexus of the postnatal retina that forms a 2D flat vascular structure that can easily be assessed, imaged and analysed, in principle even with a standard fluorescence microscope. Keeping in mind this drawback, our comparative study between the hyper- and normotensive rat strains allowed us to detect a temporal delay in the maturation of the NVU in the SHR. This observation is in agreement to the rest of the developmental parameters that were assessed in this work. We interpret that the NVU plays an active role in the establishment of the neural circuits and proper synaptogenesis, and any anomaly in the consolidation of the BBB would affect many neural functions. However, we should consider cautiously this preliminary observation and a more thoughtful analysis is required, using confocal microscopy in carefully selected equivalent areas within particular brain regions.
An interesting result was the differential characteristics of the cerebellar and cerebral cortex microvasculature. The cortical development of the rat cerebellum was divided histologically into four stages: From birth to P4 occurs an intensive proliferation of neuroprogenitor cells in the EGL. By this time, cerebellar foliation has already begun. During the third stage (from P4 to P14), the neuroblasts, which had been produced in the EGL, undergo radial migration as stellate cells, basket cells and granular cells, the latter being the most predominant. The thickness of the EGL increased most prominently around P10. In the final stage, there is an almost complete disappearance of the EGL, resulting in the cellular architecture of the adult form of the cerebellar cortex . Furthermore, these authors observed that vascular proliferation in the developing cerebellum correlated with EGL-formation pertains to the pial vessels, and not to the intraneural ones, which develop after neuronal cell migration has taken place, which would be around P21. This would explain the similar values we obtained in the microvessels analysis that were assessed before the consolidation of the cerebellar architecture, during the period of cell migration in both rat strains.
In the cerebral cortex, the comparative analysis between strains showed increased vessels length and Lacunarity index in the SHR when labelled with CAV-1 and no changes in the same vessels labelled with IB4, indicating that the microvasculature has developed in this organ, but may not have consolidated the structure of the NVU.
Microglia: Microglia in white matter regions displays unique characteristics in the first postnatal week of murine life. In both the corpus callosum and cerebellum microglia show amoeboid morphology and a similar transcription profile during development including high expression of genes related to priming of microglia, phagocytosis and migration at P7; characteristics which are already lost at P10. During the second and third week, microglia with both thick and thin processes are more abundant . White matter microglia, perhaps by migration and phagocytosis, may play a special role in local tissue dynamics around P7, impacting oligodendrocyte homeostasis and myelination. This role is likely restricted to a brief window of time, after which cell numbers decline through apoptosis. As maturation continues into adulthood, resident microglial populations develop a common homeostatic role across white and grey matter regions . Microglial cells are considered directly involved in the remodelling of neural synaptic circuits. The motor and sensorial stimulation during this developmental period may be regulating synaptic connections and microglial phagocytic activity in the cerebellum. By the second postnatal week rat pups generally open their eyes and also begin to respond to auditory signals , both neurological parameters are delayed by about one day in the SHR pups , and the presence of immature microglia at this time may correlate to the cerebellar morphological features described in this work.
Intrauterine growth restriction (IUGR) model of uterine arteries ligation cause delayed oligodendrocyte maturation and myelination accompanied by inflammatory responses in the rat . Similar situation has been described in the IU growth restricted humans . Under these circumstances, the placental insufficiency derives in chronic inflammatory processes, which may not be apparently the case of the SHR at this early postnatal period. However, around the time when hypertension signs begin to manifest, the underlying inflammatory factors may have been accumulating during an apparently silent early postnatal period. A more detailed study entailing inflammation markers assessed during the first and second postnatal weeks is still required to answer this issue.
The unfavourable uterine environment causing growth restriction results in programming that predisposes IUGR/FGR infants to long-term health issues such as poor physical growth, metabolic syndrome, cardiovascular disease, neurodevelopmental impairment and endocrine abnormalities . To find reliable biological markers for the development and evaluation of early therapeutic interventions during gestation and early postnatal life remains as a complex and challenging objective. In sight of the present results we propose to consider the developing SHR as a relevant translational animal model for developmental impairment and preliminary results from our group are showing the possibility of reversal of the negative signs (unpublished results).