Effects of extracellular calcium and surgical techniques on restoration of axonal continuity by polyethylene glycol fusion following complete cut or crush severance of rat sciatic nerves.

Complete crush or cut severance of sciatic nerve axons in rats and other mammals produces immediate loss of axonal continuity. Loss of locomotor functions subserved by those axons is restored only after months, if ever, by outgrowths regenerating at ∼1 mm/day from the proximal stumps of severed axonal segments. The distal stump of a severed axon typically begins to degenerate in 1-3 days. We recently developed a polyethylene glycol (PEG) fusion technology, consisting of sequential exposure of severed axonal ends to hypotonic Ca(2+) -free saline, methylene blue, PEG in distilled water, and finally Ca(2+) -containing isotonic saline. This study examines factors that affect the PEG fusion restoration of axonal continuity within minutes, as measured by conduction of action potentials and diffusion of an intracellular fluorescent dye across the lesion site of rat sciatic nerves completely cut or crush severed in the midthigh. Also examined are factors that affect the longer-term PEG fusion restoration of lost behavioral functions within days to weeks, as measured by the sciatic functional index. We report that exposure of cut-severed axonal ends to Ca(2+) -containing saline prior to PEG fusion and stretch/tension of proximal or distal axonal segments of cut-severed axons decrease PEG fusion success. Conversely, trimming cut-severed ends in Ca(2+) -free saline just prior to PEG fusion increases PEG fusion success. PEG fusion prevents or retards the Wallerian degeneration of cut-severed axons, as assessed by measures of axon diameter and G ratio. PEG fusion may produce a paradigm shift in the treatment of peripheral nerve injuries. © 2016 Wiley Periodicals, Inc.

Behavior and monoamine deficits in prenatal and perinatal iron deficiency are not corrected by early postnatal moderate-iron or high-iron diets in rats.

Developmental iron deficiency anemia (IDA) causes brain and behavioral deficits in rodent models, which cannot be reversed when treated at periods equivalent to later infancy in humans. This study sought to determine whether earlier iron treatment can normalize deficits of IDA in rats and what iron dose is optimal. The offspring of dams with IDA during gestation were cross-fostered at postnatal d (P) 8 to dams receiving diets with 1 of 3 iron concentrations until weaning (P21): 0.003-0.01 g/kg [totally iron deficient (TID)]; 0.04 g/kg [formerly iron deficient (FID-40)]; or 0.4 g/kg (FID-400). Always iron-sufficient control dams (CN-40) received a 0.04-g/kg iron diet. At P21, TID pups received a 0.01 g iron/kg diet; all others received a 0.04 g iron/kg diet. Hematocrit and brain iron and monoamine concentrations were assessed at P21 and P100. Pup growth, development, activity, object recognition, hesitancy, and watermaze performance were evaluated. Regional brain iron was restored by iron treatment. Regional monoamine and metabolite concentrations were elevated in FID-40 rats and reduced in FID-400 and TID rats compared with CN-40 rats. FID-40 offspring had motor delays similar to TID during lactation and FID-400 rats had elevated thigmotaxis similar to TID rats at P25 and P100 in the spatial watermaze. In conclusion, iron treatment at P8 in rats did not normalize all monoamine or behavioral measures after early IDA. Moderate iron treatment improved adult behavior, but higher iron treatment caused brain and behavioral patterns similar to TID in the short and long term.

Cranial and related sensorimotor impairments in rodent models of Parkinson's disease.

In Parkinson's disease both limb and cranial sensorimotor functions are impaired, leading to a profound diminished quality of life for many patients. Toxin and genetic animal models of Parkinson's disease are likely essential for understanding the pathology associated with these impairments as well as for the development and testing of potential therapeutics. Here we describe useful novel and established behavioral outcome measures for assessing limb and cranial sensorimotor functions in toxin and genetic models of parkinsonism in rats and mice.

Retinoic acid and environmental enrichment alter subventricular zone and striatal neurogenesis after stroke.

Neurogenesis increases in the adult rodent forebrain subventricular zone (SVZ) after experimental stroke. Newborn neurons migrate to the injured striatum, but few survive long-term and little evidence exists to suggest that they integrate or contribute to functional recovery. One potential strategy to improve stroke recovery is to stimulate neurogenesis and integration of adult-born neurons by using treatments that enhance neurogenesis. We examined the influence of retinoic acid (RA), which stimulates neonatal SVZ and adult hippocampal neurogenesis, and environmental enrichment (EE), which enhances survival of adult-born hippocampal neurons. We hypothesized that the combination of RA and EE would promote survival of adult-generated SVZ-derived neurons and improve functional recovery after stroke. Adult rats underwent middle cerebral artery occlusion, received BrdU on days 5-11 after stroke and were treated with RA/EE, RA alone, EE/vehicle or vehicle alone and were killed 61 days after stroke. Rats underwent repeated MRI and behavioral testing. We found that RA/EE treatment preserved striatal and hemisphere tissue and increased SVZ neurogenesis as demonstrated by Ki67 and doublecortin (DCx) immunolabeling. All treatments influenced the location of BrdU- and DCx-positive cells in the post-stroke striatum. RA/EE increased the number of BrdU/NeuN-positive cells in the injured striatum but did not lead to improvements in behavioral function. These results demonstrate that combined pharmacotherapy and behavioral manipulation enhances post-stroke striatal neurogenesis and decreases infarct volume without promoting detectable functional recovery. Further study of the integration of adult-born neurons in the ischemic striatum is necessary to determine their restorative potential.

Moderate iron deficiency in infancy: biology and behavior in young rats.

Iron deficiency anemia in early childhood is associated with developmental delays and perhaps, irreversible alterations in neurological functioning. The goals were to determine if dietary induced gestational and lactational iron deficiency alters brain monoamine metabolism and behaviors dependent on that neurotransmitter system. Young pregnant rats were provided iron deficient or control diets from early in gestation through to weaning of pups and brain iron concentration, regional monoamine variables and achievement of specific developmental milestones were determined throughout lactation. Despite anemia during lactation, most brain iron concentrations did not fall significantly until P25, and well after significant changes in monoamine levels, transporter levels, and D2R density changed in terminal fields. The changes in D2R density were far smaller than previously observed models that utilized severe dietary restriction during lactation or after weaning. Iron deficient pups had normal birth weight, but were delayed in the attainment of a number of milestones (bar holding, vibrissae-evoked forelimb placing). This approach of iron deficiency in utero and during lactation sufficient to cause moderate anemia but not stunt growth demonstrates that monaminergic metabolism changes occur prior to profound declines in brain iron concentration and is associated with developmental delays. Similar developmental delays in iron deficient human infants suggest to us that alterations in iron status during this developmental period likely affects developing brain monaminergic systems in these infants.

Experimental focal ischemic injury: behavior-brain interactions and issues of animal handling and housing.

In experimental neurological models of brain injury, behavioral manipulations before and after the insult can have a major impact on molecular, anatomical, and functional outcome. Investigators using animals for preclinical research should keep in mind that people with brain injury have lived in, and will continue to live in, an environment that is far more complex than that of the typical laboratory rodent. To yield more reliable and relevant behavioral assessment, it may be appropriate in some cases to house animals in environments that allow for motor enrichment and to handle animals in ways that promote tameness. Experience can affect mechanisms of plasticity and degeneration beneficially or adversely. Behavioral interventions that have been found to modulate postinjury brain events are reviewed. The timing and interaction of biological and motor therapies and the potential contribution of experience-dependent and drug-induced trophic factor expression are discussed.

Should the injured and intact hemispheres be treated differently during the early phases of physical restorative therapy in experimental stroke or parkinsonism?

Over a century ago the intact cortex was proposed to contribute to recovery from unilateral brain injury, but its possible role in functional outcome has become more appreciated in recent years as a result of anatomic, metabolic and behavioral studies. Although use of the contralesional limb is naturally impaired after sensorimotor cortex injury, neural and astrocytic events in the intact hemisphere may give rise to, and may be influenced by, an enhanced ability to compensate for lost motor function. The debate is still open as to whether the neural changes are generally compensatory in nature, with activity in the homotopic cortex leading to greater capability in the nonimpaired limb, or whether they are actually a matter of reorganization in the homotopic cortex leading to connections to denervated targets in the opposite hemisphere, thus allowing the homotopic cortex to control motor programs there. Although both phenomena may occur to some degree, there is mounting evidence in support of the former view. Careful behavioral techniques have been developed that can expose compensatory tricks, and the time course of these behaviors correlates well with anatomic data. Moreover, if the intact cortex sustains a second lesion after recovery from the first, forelimb sensorimotor function specific to the first-impaired side of the body is not worsened. Partial denervation of callosal fibers coming from the injured hemisphere, plus preferential use of the good forelimb caused by a cortical injury, may increase trophic factors in the intact hemisphere. These and related events seem to provide a growth-favorable environment there that permits motor learning in the intact forelimb at a level of skill exceeding that which a normal animal can attain in the same period of time. There are anecdotal cases in human neurologic patients that are consistent with these findings. For example, a colleague of the authors who sustained a unilateral infarction that rendered his dominant right hand severely impaired noticed that soon after the stroke he was able to use his left hand for writing and computers as well as he had ever used his right hand. Cross-midline placing tests also indicate that the structural events observed in the intact cortex may potentiate projections to the damaged hemisphere. These changes may help restore the capacity of tactile information projecting to the intact hemisphere to control limb placing in the impaired forelimb. Neural events in the injured hemisphere can be affected by behavior differently than the neural events in the intact hemisphere. Different therapeutic strategies might well be used on opposing limbs at different times after unilateral sensorimotor cortex injury to optimize recovery (and, indeed, to avoid exaggerating the insult). Finally, the details of reorganization in both hemispheres differ greatly depending on the type of brain injury sustained (eg, in stroke versus Parkinson's disease), suggesting that an approach that considers the role of both hemispheres is likely to be beneficial in research on a broad variety of brain pathologies.