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Study design:In vivo study using a moderate spinal cord contusion injury (SCI) model in adult rat.Objective:To assess the immunomodulatory effects of the purine nucleoside inosine on macrophage/microglia activation at and near the lesion site and in white matter areas remote from the injury epicenter.Setting:Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY, USA.Methods:Animals (N=56) were injured using a moderate SCI at T9–T10 spinal level and were divided into three groups, depending on treatment paradigm. Rats received either intraperitoneal or subcutaneous injections of inosine (N=28) or vehicle (N=28). Spinal cord tissue was processed for ED-1 immunoreactivity and the volume fraction of ED-1+ profiles was calculated using the Cavalieri method and unbiased stereology.Results:The volume fraction of ED-1+ profiles within gray and lateral white matter regions at and around the lesion site was significantly reduced only following a twice daily-6 week treatment course, compared with vehicle controls, and white matter areas remote from the lesion were unaffected by all inosine treatment paradigms.Conclusions:Continued subcutaneous delivery of inosine, beginning 15-min post-SCI and persisting throughout the survival period of 6 weeks exerted immunomodulatory effects at and around the lesion site.Spinal Cord (2008) 46, 39–44; doi:10.1038/sj.sc.3102057; published online 3 April 2007 [ABSTRACT FROM AUTHOR]

Abstract Background Propriospinal neurons, with axonal projections intrinsic to the spinal cord, have shown a greater regenerative response than supraspinal neurons after axotomy due to spinal cord injury (SCI). Our previous work focused on the response of axotomized short thoracic propriospinal (TPS) neurons following a low thoracic SCI (T9 spinal transection or moderate spinal contusion injury) in the rat. The present investigation analyzes the intrinsic response of cervical propriospinal neurons having long descending axons which project into the lumbosacral enlargement, long descending propriospinal tract (LDPT) axons. These neurons also were axotomized by T9 spinal injury in the same animals used in our previous study. Results Utilizing laser microdissection (LMD), qRT-PCR, and immunohistochemistry, we studied LDPT neurons (located in the C5-C6 spinal segments) between 3-days, and 1-month following a low thoracic (T9) spinal cord injury. We examined the response of 89 genes related to growth factors, cell surface receptors, apoptosis, axonal regeneration, and neuroprotection/cell survival. We found a strong and significant down-regulation of ~25% of the genes analyzed early after injury (3-days post-injury) with a sustained down-regulation in most instances. In the few genes that were up-regulated (Actb, Atf3, Frs2, Hspb1, Nrap, Stat1) post-axotomy, the expression for all but one was down-regulated by 2-weeks post-injury. We also compared the uninjured TPS control neurons to the uninjured LDPT neurons used in this experiment for phenotypic differences between these two subpopulations of propriospinal neurons. We found significant differences in expression in 37 of the 84 genes examined between these two subpopulations of propriospinal neurons with LDPT neurons exhibiting a significantly higher base line expression for all but 3 of these genes compared to TPS neurons. Conclusions Taken collectively these data indicate a broad overall down-regulation in the genes examined, including genes for neurotrophic/growth factor receptors as well as for several growth factors. There was a lack of a significant regenerative response, with the exception of an up-regulation of Atf3 and early up-regulation of Hspb1 (Hsp27), both involved in cell stress/neuroprotection as well as axonal regeneration. There was no indication of a cell death response over the first month post-injury. In addition, there appear to be significant phenotypic differences between uninjured TPS and LDPT neurons, which may partly account for the differences observed in their post-axotomy responses. The findings in this current study stand in stark contrast to the findings from our previous work on TPS neurons. This suggests that different approaches will be needed to enhance the capacity for each population of propriospinal neuron to survive and undergo successful axonal regeneration after SCI.

Abstract Background Central nervous system axons lack a robust regenerative response following spinal cord injury (SCI) and regeneration is usually abortive. Supraspinal pathways, which are the most commonly studied for their regenerative potential, demonstrate a limited regenerative ability. On the other hand, propriospinal (PS) neurons, with axons intrinsic to the spinal cord, have shown a greater regenerative response than their supraspinal counterparts, but remain relatively understudied in regards to spinal cord injury. Results Utilizing laser microdissection, gene-microarray, qRT-PCR, and immunohistochemistry, we focused on the intrinsic post-axotomy response of specifically labelled thoracic propriospinal neurons at periods from 3-days to 1-month following T9 spinal cord injury. We found a strong and early (3-days post injury, p.i) upregulation in the expression of genes involved in the immune/inflammatory response that returned towards normal by 1-week p.i. In addition, several regeneration associated and cell survival/neuroprotective genes were significantly up-regulated at the earliest p.i. period studied. Significant upregulation of several growth factor receptor genes (GFRa1, Ret, Lifr) also occurred only during the initial period examined. The expression of a number of pro-apoptotic genes up-regulated at 3-days p.i. suggest that changes in gene expression after this period may have resulted from analyzing surviving TPS neurons after the cell death of the remainder of the axotomized TPS neuronal population. Conclusions Taken collectively these data demonstrate that thoracic propriospinal (TPS) neurons mount a very dynamic response following low thoracic axotomy that includes a strong regenerative response, but also results in the cell death of many axotomized TPS neurons in the first week after spinal cord injury. These data also suggest that the immune/inflammatory response may have an important role in mediating the early strong regenerative response, as well as the apoptotic response, since expression of all of three classes of gene are up-regulated only during the initial period examined, 3-days post-SCI. The up-regulation in the expression of genes for several growth factor receptors during the first week post-SCI also suggest that administration of these factors may protect TPS neurons from cell death and maintain a regenerative response, but only if given during the early period after injury.

III-V semiconductor nanowire (NW) heterostructures with axial InGaAs active regions hold large potential for diverse on-chip device applications, including site-selectively integrated quantum light sources, NW lasers with high material gain, as well as resonant tunneling diodes and avalanche photodiodes. Despite various promising efforts toward high-quality single or multiple axial InGaAs heterostacks using noncatalytic growth mechanisms, the important roles of facet-dependent shape evolution, crystal defects, and the applicability to more universal growth schemes have remained elusive. Here, we report the growth of optically active InGaAs axial NW heterostructures via completely catalyst-free, selective-area molecular beam epitaxy directly on silicon (Si) using GaAs(Sb) NW arrays as tunable, high-uniformity growth templates and highlight fundamental relationships between structural, morphological, and optical properties of the InGaAs region. Structural, compositional, and 3D-tomographic characterizations affirm the desired directional growth along the NW axis with no radial growth observed. Clearly distinct luminescence from the InGaAs active region is demonstrated, where tunable array-geometry parameters and In content up to 20% are further investigated. Based on the underlying twin-induced growth mode, we further describe the facet-dependent shape and interface evolution of the InGaAs segment and its direct correlation with emission energy., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The glial scar formed at the site of traumatic spinal cord injury (SCI) has been classically hypothesized to be a potent physical and biochemical barrier to nerve regeneration. One longstanding hypothesis is that the scar acts as a physical barrier due to its increased stiffness in comparison to uninjured spinal cord tissue. However, the information regarding the mechanical properties of the glial scar in the current literature is mostly anecdotal and not well quantified. We monitored the mechanical relaxation behavior of injured rat spinal cord tissue at the site of mid-thoracic spinal hemisection 2 weeks and 8 weeks post-injury using a microindentation test method. Elastic moduli were calculated and a modified standard linear model (mSLM) was fit to the data to estimate the relaxation time constant and viscosity. The SLM was modified to account for a spectrum of relaxation times, a phenomenon common to biological tissues, by incorporating a stretched exponential term. Injured tissue exhibited significantly lower stiffness and elastic modulus in comparison to uninjured control tissue, and the results from the model parameters indicated that the relaxation time constant and viscosity of injured tissue were significantly higher than controls. This study presents direct micromechanical measurements of injured spinal cord tissue post-injury. The results of this study show that the injured spinal tissue displays complex viscoelastic behavior, likely indicating changes in tissue permeability and diffusivity.

Previous studies show that most short thoracic propriospinal (TPS; T5-T7) and long descending propriospinal tract (LDPT; C4-C6) neurons are lost following low-thoracic spinal cord contusion injury (cSCI), as assessed by retrograde labeling with fluorogold (FG). Gene microarray and terminal deoxynucleotidyl transferase dUTP nick end (TUNEL)/caspase-3 immunolabeling indicate that post-axotomy cell death may be responsible for the observed decrease in number of labeled TPS neurons post cSCI. Yet, no indications of post-axotomy cell death are evident within LDPT neurons following the same injury. The present experiments were devised to understand this difference. We assessed the number and size of LDPT and TPS neurons at different time points, retrogradely labeling these neurons with FG prior to delivering a moderate low-thoracic cSCI or after they were axotomized by a complete low-thoracic spinal transection. Counts of FG-filled TPS and LDPT cells indicate a large loss of both neuronal populations 2 weeks post cSCI. Propriospinal neurons in other animals were retrogradely labeled with dextran tetramethyl rhodamine prior to cSCI and tissue was processed for detection of TUNEL- or caspase-3-positive profiles at chronic times post injury. Our overall findings confirm that cell death post injury is the major factor responsible for the loss of TPS neurons during the acute period post cSCI, and that little post-axotomy cell death occurs in LDPT neurons during the first 2 months after the same injury. After chronic axotomy retrograde transport is impaired in LDPT neurons, but can be reinitiated by re-axotomy. Our results also indicate that FG is metabolized/lost from retrogradely labeled neurons at increasing survival times, and that this process appears to be accelerated by injury., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Raman spectroscopy was used to study temporal molecular changes associated with spinal cord injury (SCI) in a rat model. Raman spectra of saline-perfused, injured, and healthy rat spinal cords were obtained and compared. Two injury models, a lateral hemisection and a moderate contusion were investigated. The net fluorescence and the Raman spectra showed clear differences between the injured and healthy spinal cords. Based on extensive histological and biochemical characterization of SCI available in the literature, these differences were hypothesized to be due to cell death, demyelination, and changes in the extracellular matrix composition, such as increased expression of proteoglycans and hyaluronic acid, at the site of injury where the glial scar forms. Further, analysis of difference spectra indicated the presence of carbonyl containing compounds, hypothesized to be products of lipid peroxidation and acid catalyzed hydrolysis of glycosaminoglycan moieties. These results compared well with in vitro experiments conducted on chondroitin sulfate sugars. Since the glial scar is thought to be a potent biochemical barrier to nerve regeneration, this observation suggests the possibility of using near infrared Raman spectroscopy to study injury progression and explore potential treatments ex vivo, and ultimately monitor potential remedial treatments within the spinal cord in vivo.

We studied the number, location and size of long descending propriospinal tract neurons (LDPT), located in the cervical enlargement (C3-C6 spinal levels), and short thoracic propriospinal neurons (TPS), located in mid-thoracic spinal cord (T5-T7 spinal levels), 2, 6 and 16 weeks following a moderate low thoracic (T9) spinal cord contusion injury (SCI; 25 mm weight drop) and subsequent injections of fluorogold into the upper lumbosacral enlargement (L2-L4 spinal levels). Retrograde labeling showed that approximately 23% of LDPT and 10% of TPS neurons were labeled 2 weeks after SCI, relative to uninjured animals. No additional significant decrease in number of labeled LDPT and TPS cells was found at the later time points examined, indicating that the maximal loss of propriospinal neurons in these two subpopulations occurs within the first 2 weeks post-SCI. The distribution of labeled cells post-moderate SCI was similar to normal in terms of their location within the gray matter. However, there was a significant change in the size (cross sectional area) of labeled neurons following injury, relative to uninjured controls, indicating a loss in the number of the largest class of propriospinal neurons. Interestingly, the number of labeled LDPT and TPS neurons was not significantly different following different injury severities. Although the rostro-caudal extent of the lesion site expanded between 2 and 16 weeks following injury, there was no significant difference in the number of propriospinal neurons that could be retrogradely labeled at these time points. Possible reasons for these findings are discussed., (Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

The authors relate their experiences with interprofessional teaching of ethics at the University of Colorado Health Sciences Center, describing the history, planning, content, and structure of a required ethics course and discussing its role in the institution's plan to create more interprofessional education opportunities. The authors describe both the benefits of teaching ethics on an interprofessional basis and the challenges they encountered in launching the course. Challenges included responding to diverse and divergent faculty and student reactions, controlling a curriculum across schools, and learning how to think about education in interprofessional rather than profession-specific ways. Included in the discussion are the results obtained with various evaluation tools designed and assessed by the Office of Education on the campus, to which students and faculty responded the first time the course was offered.

The response to injury of ascending collaterals of dorsal root axons within the dorsal column (DC) was studied after neonatal spinal overhemisection (OH) made at different levels of the spinal cord. The transganglionic tracer, cholera toxin conjugated to horseradish peroxidase, and the anterograde tracer, biotinylated dextran amine, were used to label dorsal root ganglion cells with peripheral axons contributing to the sciatic nerve. There was no indication of a regenerative attempt by DC axons at acute survival times (3 days and later) after cervical injury, replicating previous work done at chronic survival periods (Lahr and Stelzner [1990] J. Comp. Neurol. 293:377-398). There was also no evidence of DC regeneration after lumbar OH injury even though immunohistochemical studies using the oligodendrocyte markers Rip and myelin basic protein showed few oligodendrocytes in the gracile fasciculus at lumbar levels at birth. Therefore, the lack of myelin in the dorsal funiculus at lumbar levels does not enhance the growth of neonatally axotomized DC axons. In addition, DC axons did not regenerate when presented with fetal spinal tissue implanted into thoracic OH lesions, even though positive control experiments showed that segmental dorsal root axons containing calcition gene-related peptide and corticospinal axons grew into these implants, replicating previous work of others. When a thoracic OH lesion, with or without a fetal spinal implant, was combined with sciatic nerve injury to attempt to stimulate an intracellular regenerative response of DRG neurons, again, no evidence of DC axonal regeneration was detected. Quantitative studies of the L4 and L5 dorsal root ganglia (DRG) showed that OH injury did not result in DRG neuronal loss. However, sciatic nerve injury did result in significant post-axotomy retrograde cell loss of DRG neurons, even in groups receiving thoracic embryonic spinal implants, and is one explanation for the minimal effect of sciatic nerve injury on DC regeneration. Although fetal tissue did not appear to rescue a significant number of DRG neurons, the quantitative analysis showed an enlargement of the largest class of DRG neuron, the class that contributes to the DC projection, in all groups receiving fetal tissue implants. This apparent trophic effect did not affect DC regeneration or neuronal survival after peripheral axotomy. Further studies are needed to determine why DC axons do not regenerate in a neonatal spinal environment or within fetal tissue implants, especially because previous work by others in both the developing and adult spinal cord shows that dorsal root axons will grow within the same type of fetal spinal implant.

Hindlimb weight support and bipedal stepping occur after spinal cord transection in neonatal rats (birth to 12 days of age) while the same lesion in 15-day and older animals results in permanent loss of these responses. Some compensatory change in lumbar spinal circuitry must occur after spinal transection in young animals subserving these hindlimb behaviors. In contrast, animals just a few days older are incapable of such compensatory responses. We have examined the hypothesis that neural activity leads to the postnatal loss of plasticity in spinal circuitry. We find that antagonism of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor with MK-801 in young animals extends the sparing of hindlimb function after spinal transection to older animals. This effect is not due to a non-specific depression of all exciatory drive to motor neurons since Ia to motor neurons synaptic transmission through non-NMDA receptors is preserved during MK-801 treatment. Acute administration of MK-801 at the time of spinal transection or chronic administration of MK-801 after postnatal day 17 has no effect on recovery of hindlimb function after spinal transection. These results highlight the importance of NMDA receptor activation in spinal circuit maturation.

It has been established that neonatal corticospinal (CS) axons are able to grow around lesions of the spinal cord early in neonacy (Bernstein and Stelzner, J. Comp. Neurol. 221:382-400; Firkins, Bates, and Stelzner, Exp. Neurol., 120:1-15). To determine if these corticospinal axons include regenerating as well as late developing axons a double-labeling paradigm is used in which CS neurons are retrogradely labeled from the cervical spinal cord by injections of fast blue (FB) on Postnatal Day (PND) 2, 4, or 10. Two days later, the FB is aspirated along with the left dorsal funiculus and the right hemicord (CHR). As adults, the animals receive an injection of diamidino yellow (DY) or rhodamine into the spinal cord caudal to the lesion site. Thus, FB neurons are those that originally projected to the spinal cord before the lesion and which survived axotomy, DY neurons are those whose axons reached the spinal cord after the lesion, and double-labeled neurons (FB/DY) are cells which projected to the spinal cord prior to the lesion and regenerated a spinal axon postlesion. Animals FB injected on PND 2 have a widespread distribution of FB-labeled neurons in cortex, including areas outside of sensorimotor cortex. These animals also had both DY- and FB/DY-labeled cells within sensorimotor cortex, indicating that the population of axons growing caudal to neonatal spinal lesions consists of both late growing and regenerating axons. In animals FB injected on PND 10, the FB neurons were all located in sensorimotor cortex. Very few DY and no FB/DY neurons were present. We have also looked at the topography of the CS neurons which project caudal to early spinal lesions. Rat pups received a CHR on PND 0, 3, 6, or 12. As adults, horseradish peroxidase was injected into the cervical or lumbar enlargement of the spinal cord and the distribution of labeled cells in the cerebral cortex was plotted and compared to normal and lesioned adult controls. In all experimental animals, the distribution of retrogradely labeled cells was restricted to the area containing CS projection neurons in the normal animal. This is despite the fact that the number of CS projection neurons is greatly reduced from normal and the normal pathway for CS axonal outgrowth has been completely disrupted by the neonatal lesion.

We have investigated corticospinal (CS) axon growth around cervical spinal injury in the neonatal rat and related this growth to the astroglial reaction occurring at the lesion site. Rats received a high cervical overhemisection (left dorsal funiculotomy, right spinal hemisection) and a right cortical ablation on Postnatal Days (PNDs) 0, 3, 6, 12, and 21 to 24 (weanlings). In chronic operates the remaining CS projection from the left sensorimotor cortex was then assayed using wheat germ agglutinin-horseradish peroxidase as an anterograde tracer. In other operates the formation of the astroglial scar at the spinal lesion site was studied using a monoclonal antibody to glial fibrillary acidic protein. In PNDs 0-6 operates labeled axons extend through the intact left hemicord to bypass the lesion. The labeled axons travel to the edge of the lesion, cross the midline, and pass lateral to the lesion within the dorsal and intermediate gray and dorsal lateral white matter. Axons project bilaterally to normal areas of CS termination in PND 0 operates for a distance of 2.5 to 4 spinal segments caudal to the lesion which decreases to 1.5 to 2 segments in PND 6 operates. In PND 12 and weanling operates labeled fibers do not grow around the lesion but instead are retracted rostrally. There is an astrocytic reaction to injury at all ages by 3 days postoperatively (p.o.) that becomes greater with age and p.o. survival time. A more complicated cystic scar forms in 6-day and older operates. These data show that there is an age-related change in the ability of CS axons to grow around spinal injury which ends near the time CS elongation and gliogenesis is complete in the spinal cord.

Lateral hemisection lesions separated by 1 to 3 spinal segments were made on opposite sides of the mithoracic spinal cord in 1-month-old (N = 15; weanling operates) and newborn albino rats (N = 16; neonatal operates). Hindlimb behavior was assessed between 1 and 6 months p.o. for both groups of operates using a protocol and rating system that have previously proved effective in differentiating behavioral recovery of the hindlimbs as a function of age of spinal transection. In addition, at the conclusion of behavioral testing, operates received spinal injections of [3H]proline and HRP caudal to the spinal lesions to determine if lesions were complete and if neurons within the region between the two lesions (interlesion zone) projected into the caudal spinal cord. In both groups of operates, neurons were retrogradely labeled within the interlesion zone bilaterally, primarily in laminae VII-VIII. When both lesions were complete lateral hemisections in weanling operates, little behavioral recovery was observed, similar to complete spinal cord transection (N = 3). However, much greater behavioral recovery was seen, including supporting reactions and locomotor responses, when one or both lesions spared axons along the ventrolateral rim of the white matter. Neurons were retrogradely labeled in the brain stem reticular formation (N = 12) in these cases. All lesions were complete lateral hemisections in neonatal operates but much greater behavioral recovery was seen than in weanling operates with the same lesions, including supporting, placing, and locomotor responses. In an additional group of eight neonatal operates, the spinal cord rostral to the spinal hemisections was transected at 1 month of age. Supportive, placing, and locomotor responses were seen immediately after recovery from anesthesia and responses returned to pretransection levels in six of eight operates over the 10-day survival period. Fink-Heimer impregnation showed that degeneration argyrophilia from the transection bilaterally filled the interlesion zone but little argyrophilia was seen caudal to this region. Our results indicate that an intact propriospinal circuit remains in both neonatal and weanling operates but does not appear to contribute to hindlimb response development or recovery. The greater behavioral recovery in neonatal operates appears due to intrinsic connections (doral root, interneuronal) continuing to be able to drive the spinal circuitry underlying the spared behaviors.

Tectal efferent axons, located adjacent to the optic tract, fail to regenerate past diencephalic lesions in Rana pipiens even though optic axons regenerate after the same injury (M. J. Lyon and D. J. Stelzner, J. Comp. Neurol. 255: 511-525). We tested the possibility that tectal efferent axons can regenerate within peripheral nerve implants. A 6- to 8-mm segment of autologous sciatic nerve was implanted into the anterolateral (N = 23) or centrolateral (N = 22) portion of the dorsal surface of the tectum. Frogs survived for 6 (N = 16) or 12 weeks (N = 29) before the free end of the nerve was recut and HRP applied. A control group had the nerve crushed prior to the HRP application. Neurons within the tectum, near and medial to the implant site, were retrogradely labeled from the nerve graft in most experimental operates but no neurons were labeled in controls. In addition, neurons were also labeled in nuclei which projected to the tectum in a number of cases. Three times as many neurons were labeled in 12-week operates (42 +/- 46) as in 6-week operates (15 +/- 12). The morphology and location of labeled neurons in the tectum was similar to tectal efferent neurons except that the somal area of neurons labeled from the graft was significantly larger (41%) than normal tectal efferent neurons. The basic finding is similar to experiments using the same paradigm in the mammalian central nervous system (CNS). One difference is the minimal glial reaction at the graft insertion site.(ABSTRACT TRUNCATED AT 250 WORDS)

The response of dorsal column axons was studied after neonatal spinal overhemisection injury (right hemicord and left doral funiculus). Rat pups (N = 11) received this spinal lesion at the C2 level within 30 hours after birth. The cauda equina was exposed 3 months later in one group of chronic operates (N = 5) and in a group of normal adults (N = 2), and all spinal roots from L5 caudally were cut bilaterally; 4 days later the spinal cord and medulla were processed for Fink-Heimer impregnation of degenerating axons and terminals. In a second group of chronic operates (N = 6) and normal adult controls (N = 4) the left sciatic nerve was injected with a cholera toxin-HRP conjugate (C-HRP), followed by a 2-3 day transganglionic transport period, and then the spinal cord and medulla were processed with tetramethylbenzidine histochemistry. Both control groups have a consistent dense projection in topographically adjacent regions of the dorsal funiculus and gracile nucleus. However, there is no sign of axonal growth around the lesion in either group of chronic experimental operates. Instead, there is a decreased density of projection within the dorsal funiculus near the lesion site. Many remaining C-HRP labeled axons in the experimental operates have abnormal, thick varicosities and swollen axonal endings (5-10 microns x 10-30 microns) within the dorsal funiculus through several spinal segments caudal to the lesion. Ultrastructural analysis of the dorsal funiculus in three other chronic experimental operates reveals the presence of numerous vesicle filled axonal profiles and reactive endings which appear similar to the C-HRP labeled structures. Transganglionic labeling after C-HRP sciatic nerve injections (N = 4) and retrograde labeling of L4, L5 dorsal root ganglion neurons after fast blue injections of the gracile nucleus (N = 6) both suggest that all dorsal column axons project to the gracile nucleus in the newborn rat. Dorsal root ganglion (DRG) cell survival following the neonatal overhemisection injury was also examined in the L4 and L5 DRG. DRG neurons that project to the gracile nucleus were prelabeled by injecting fast blue into this nucleus at birth two days prior to the cervical overhemisection spinal injury. Both normal littermates (N = 9) and spinally injured animals (N = 12) were examined after postinjection survival periods of 10 or 22 days.(ABSTRACT TRUNCATED AT 400 WORDS)

The spinal cord of neonatal and weanling rats was mid-thoracically transected. Either 3 or 6 months later the borders of the lesion site were studied using electron microscopy. No sign of axonal regeneration through the lesion site was found in either group, even though the glial reaction was minimal in neonatal operates. In both groups of operates, reactive axonal endings, presumed to result from the original surgery, and neuritic growth were found in a reactive zone on both sides of the lesion site. We conclude that the potential for axonal growth (regeneration or generation) is maintained at the borders of the lesion in both groups of operates.

In the dorsal lateral geniculate nucleus (LGN) of the adult cat there is no evidence for translaminar sprouting of retinal axons to fill sites freed of retinal endings from the other eye. We tested the possibility that retinal axons will sprout to fill denervated retinal sites within laminae of the monkey LGN. In 4 monkeys, retinal ganglion cell axons from either the upper or lower half of the retina were destroyed. To maximize the potential for sprouting in the LGN, on one side of the brain the LGN cells to which the remaining retinal axons normally project were removed by ablation of the appropriate portion of the striate cortex. Three months later the eye receiving the retinal lesion was injected with [3H]proline and the retinal projection to the LGN on both sides of the brain was studied using autoradiography. We found no evidence of intralaminar sprouting of retinal axons either in the normal LGN or in the LGN in which the usual targets of retinal axons had been removed.

The glycoprotein laminin is a prominent constituent of basal laminae and has been suggested to play an important role in axonal growth. We have tested this hypothesis, by examining the temporal and spatial distribution of laminin in the rat spinal cord, relative to elongating corticospinal tract (CST) axons, during normal development and after newborn and adult spinal lesions. The distribution of laminin was demonstrated in spinal cord sections from animals ranging in age from 14 days embryonic to adult using immunocytochemistry. Anti-laminin immunolabeling was seen around blood vessels and meninges in all the animals examined. However, within the grey and white matter its distribution was age-dependent. In the normal cord, immunostaining appeared in small amounts in early embryos, but was absent from all postnatal animals even at ages when the CST was growing down the cord. Following injury, intense immunostaining was associated with lesions in both newborn and adult operates at all postoperative periods examined. Within the matrix of the lesion laminin immunostaining was especially prominent. In the intact cord it was prominent only around blood vessels near the lesion site. Our results indicate that the distribution of laminin does not closely correlate with axonal growth of the CST either during normal development or after spinal injury.

Developing and regenerating frog optic axons grow within optic pathways and form connections only with optic targets. However, unlike normal development, many regenerating optic axons in the adult frog are misrouted within optic pathways, including axons that grow into the opposite retina. Many of the axons misrouted during regeneration appear to be collaterals of axons that grow in normal directions. Ganglion cell loss of up to 60% occurs after optic nerve damage, beginning prior to reinnervation of optic targets. Massive axonal collateralization also takes place near the point of nerve damage, causing the normal order found within the nerve to be lost. Collaterals are eliminated as selective reinnervation is completed, and the smaller complement of optic cell axons remaining after regeneration form an expanded projection within optic targets. Evidence is reviewed that suggests that factors involved in axonal guidance and target recognition during development remain intact in the adult frog brain. Additional conditions resulting from nerve injury causes axonal guidance to be less successful during regeneration.

The posterior limb of the rat anterior commissure (ACp) was studied in order to better define a temporal cortex in this species. Two methods were used: (1) the anterior commissure (AC) was destroyed on one side of the midline, and the resulting anterograde degeneration was traced with the Fink-Heimer stain; and (2) the corpus callosum and hippocampal commissure were severed, but the AC was left intact. [3H]-Leucine and proline were injected into one hemisphere, and the transported label was traced into the contralateral hemisphere with autoradiography. ACp was found to project to the contralateral cortex along the rhinal sulcus. The pyriform cortex received a projection along the entire length of the sulcus. There was also a distinct projection to neocortex on the lateral surface above the rhinal sulcus which appears to be analogous to the temporal cortex projection of ACp in other species. This finding of a neocortical projection of the ACp in the rat is consistent with observations that have been made on other mammals.

The midthoracic spinal cord was transected in neonatal (N = 12), weanling (N = 9), and adult (N = 9) rats and sections were prepared at various postoperative survival periods (2 to 106 days p.o.) using a modified Golgi-Cox stain. The morphology of large lamina IX "motoneurons" was examined 0 to 5 mm rostral to the lesion border and compared with the morphology of similar cells from this region in developing and adult rats. Motoneurons examined in neonatal operates underwent the same temporal sequence of morphologic changes seen during normal development. Motoneurons in weanling operates underwent rapid morphologic changes which were most marked at the earliest period studied (2 days p.o.). These changes included a decrease in perikaryal diameter, dendritic loss, and the appearance of perikaryal and dendritic spines and varicose swellings. At the longest p.o. survival period analyzed (60 days p.o.), neurons had partially recovered but still retained somatic and dendritic spines and a reduced dendritic arbor. Motoneurons stained in adult operates showed no morphologic changes at 15 days p.o. but by 90 days p.o. somatic and dentritic spines were apparent, more similar to the morphology normally seen in the early postnatal period. We interpret these data to indicate that motoneurons surviving spinal transection are relatively unaffected by this lesion during the synaptogenic period and most severely affected when denervated near the end of this time. The more gradual appearance of regressive morphologic changes seen in adult motoneurons may indicate the continued partial denervation of these cells.

Transecting the thoracic spinal cord of the rat has markedly different effects on behavioral responses of the hindlimbs if the lesion is made at the neonatal or weanling stage of development. The present investigation tested the possibility that the behavioral differences were related to a difference in the distribution or density of dorsal root connections in the lumbosacral spinal cord. In order to use each animal as its own control the distribution and density of dorsal root axons was compared on the two sides of the L5-S1 segments of the lumbosacral spinal cord in adult rats given a mid-thoracic spinal hemisection at the neonatal or weanling stage of development. Comparing the experimental (initially hemisected side) and control sides of the cord, we found no evidence for a change in the distribution of dorsal root axons. The distribution of Fink-Heimer stained degeneration 4--6 days after bilateral spinal root section was virtually identical on the two sides of the cord from animals hemisected at either stage. However, in rats spinally hemisected at the neonatal stage (n = 8), a significantly greater density of dorsal root degeneration was found within the intermediate nucleus of Cajal (INC) on the experimental side using coded material and a blind analysis. No difference in the density of dorsal root degeneration was detected in the group of rats spinally hemisected at the weanling stage (n = 6). Controls indicated that the increased density of degeneration was not due to compression resulting from shrinkage of the INC or to degeneration remaining from the initial hemisection. We conclude that the increased amount of argyrophilia within the INC of neonatally hemisected rats is due to an increased density of dorsal root axons in this zone. This result supports the hypothesis that the behavioral differences found when comparing animals transected at the neonatal or weanling stages of development are related to an increased number of dorsal root connections within the lumbosacral spinal cord.

Rats received a midthoracic spinal cord "overhemisection" including right hemicord and left dorsal funiculus at birth (neonatal operates, N = 15) or 21 days of age (weanling operates, N = 14). In a second experiment neonatal (N = 6), 6-day (N = 3), and 12-day (N = 7) rats sustained a right sensorimotor cortex (SmI) ablation to destroy the left corticospinal tract (CST) at the same time as the spinal injury (double lesion operates). Later (3-12 months) injections of 3H-proline and autoradiography were used to label the left or right CST. The results of the first experiment showed that most right CST axons failed to grow around the spinal lesion in neonatal operates (N = 9). There was an increase in the density of label, mainly to CST projection areas, in a 1-mm zone rostral to the lesion. However, left CST axons bypassed the lesion by growing through the intact tissue in neonatal operates (N = 6). These displaced axons were consistently located within the dorsal portion of the lateral funiculus (dLF) and remained within that location caudal to the lesion, an area normally containing only a few CST axons. In spite of this abnormal position, these axons terminated bilaterally throughout the remainder of the cord in normal CST sites. In weanling operates, CST axons severed by the lesion did not regenerate around the lesion site. An increased density of label over the few spared axons within the left dLF and in CST projection zones immediately caudal to the lesion site suggested axonal sprouting by these axons. The results of the second experiment showed that the lack of growth of right CST axons around this injury in neonatal operates was, at least partially, due to an interaction with left CST axons. In neonatal double lesion operates, right CST axons grew around the spinal injury for a varying distance within the left dLF and distributed bilaterally to normal CST sites. The number of right CST axons bypassing the lesion was related to the configuration of the lesion site. A smaller number of right CST axons bypassed the lesion in 6-day double lesion operates and most terminated within 2-3 mm of the lesion site. Right CST axons failed to grow around this injury in 12-day double lesion operates.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous reports from this laboratory have shown that a substantial number of optic axons are misrouted after optic nerve regeneration in the adult frog, Rana pipiens. Regenerating axons from a crushed optic nerve are distributed throughout the opposite nerve. In this study, we report the effect of crushing both optic nerves (double crush) on the pattern and degree of axonal misrouting. In 28 frogs both optic nerves were crushed at the same time (simultaneous double crush) and animals survived for varying periods before the right eye was injected with 3H-proline and the brain processed for autoradiography 24 hours later. In every frog with postoperative survivals longer than 2 weeks, labeled axons from the right eye were found in the left optic nerve. However, when compared to the amount of label seen in frogs in which only the right optic nerve was crushed (single crush) there was substantially less label within the left nerve of frogs after crushing both nerves. Label was also found only at the edge of the left nerve in material from double crush frogs, unlike that found after single crush. In four of six frogs where the left nerve was crushed 1 week after the right nerve (delayed double crush), the proximal end of the left nerve was completely filled with label, but more distally, label was found only along the edge of this nerve. Although fewer optic axons were labeled in the opposite optic nerve of double crush frogs, label did extend to the optic disc of that eye. However, label was not apparent in the ganglion cell fiber layer of the opposite eye. Instead, it was confined to the edge of the optic disc in a space apparently associated with papilledema resulting from crushing the optic nerve of that eye. In six frogs the retina of the left eye was removed at the same time the right optic nerve was crushed. Labeled axons of the right eye filled the left optic nerve to the retina-less shell of the left eye. Thus, these data show that the amount and distribution of axonal misrouting into the opposite optic nerve during optic nerve regeneration is affected by intact or regenerating optic axons from the other eye.

Experiments were designed to determine if neurons of the ranid optic tectum, a major target of the optic nerve, possess the same regenerative potential as optic axons. Normal tectal efferent (TE) projections were reexamined by using the anterograde transport of 3H-proline and autoradiography (n = 18), bulk-filling damaged TE axons with horseradish peroxidase (HRP; n = 18) and anterogradely transporting wheat germ agglutinin-HRP (n = 8) to label TE axons. Results were similar to reports that used degeneration methods (Rubinson: Brain Behav. Evol. 1:529-561, '68; Lazar: Acta. Biol. Hung. 20:171-183, '69). Following a brainstem hemisection just caudal to the nucleus isthmi (1-20 weeks), the ipsilateral descending TE pathway was autoradiographically examined (n = 20). While all other TE projections appeared normal, there was no detectable ipsilateral descending projection beyond the lesion site. Ascending TE axons were cut at the anterior tectal border by hemisecting the left diencephalon (LDH)--a lesion that also cuts optic axons projecting to the left tectum. There was no indication of TE axonal regeneration with the aid of autoradiography or HRP histochemistry 1-30 weeks postlesion (n = 48) even when the medial diencephalon was intentionally left intact (n = 4). However, in all four cases examined, optic axons regenerated following the same LDH where TE axonal regeneration failed (also see Stelzner, Lyon, and Strauss: Anat. Rec. 205:191A-192A, '83). Local effects of LDH should be similar for both the cut optic and cut TE axons. Other factors were tested that may contribute to the lack of TE axonal regeneration. Our results indicate that optic regeneration itself (n = 8), postaxotomy retrograde cell death of TE neurons (n = 6), deafferentation of the tectum of optic axons, and potential sprouting within tectal targets by intact contralateral TE axons (n = 10) are not critical factors aborting TE axonal regeneration. TE axons filled with HRP at chronic periods after LDH (n = 4) terminate anomalously near the LDH border. Many of these endings are similar to reactive endings or terminal clubs seen after axonal injury in the mammalian CNS. Our results suggest that this disparity in regenerative ability of optic and TE axons may be related to a difference in the responsive ability of these cell types to initiate or maintain axonal elongation after axotomy within the amphibian CNS environment.

We have reported previously that during optic nerve regeneration in Rana pipiens, axons are misrouted into the opposite nerve and retina. In the present investigation we have examined the time course of formation of these "misrouted" axons and their cells of origin. The right eye of 31 frogs was injected with 3H-proline at various times after right optic nerve crush. In every frog examined 2 weeks and later after nerve crush, the distribution of autoradiographic label indicated that axons from the right eye had grown into the left optic nerve at the chiasm. The amount of label increased from 2 weeks to reach a maximum at 6 weeks where the entire left nerve was filled with silver grains. At 5 to 6 weeks after crush, labeled axons were found within the ganglion cell fiber layer (GCFL) of the retina of the opposite eye for a maximum distance of 2.3 mm from the optic disc. In frogs examined at intervals later than 6 weeks after crush, the amount of label within the left eye and nerve progressively decreased, indicating a gradual disappearance of the misrouted axons. Studies using anterograde transport of horseradish peroxidase (HRP) after nerve injection confirmed these autoradiographic findings. The position of ganglion cells in the right eye whose axons were misrouted to the left eye was determined by retrograde transport of HRP. Five or 6 weeks after crushing the right optic nerve, the left eye was injected with HRP and labeled ganglion cells were found throughout the right eye retina. The largest percentage of labeled cells was found within the ventral half of the retina, particularly within the temporal quadrant, and nearly all of the labeled cells were found in more peripheral portions of the retina. Since few retino-retinal axons are found during normal development, the present results show that the factors guiding regenerating axons in the adult frog differ substantially from those present during development.

The spinal cords of newborn (0-3 day old) and adult rats were mid-thoracically hemisected. Ninety days later a glial and connective tissue scar had formed at the lesion site in the adult hemisected rats while in neonatally lesioned animals only normal appearing regions of the contralateral spinal cord were found in the area of hemisection. Comparisons of the magnocellular portions of the red nucleus (MPRN) revealed a decrease in cell number in the MPRN contralateral (C-MPRN) to the spinal lesion. However, only in the newborn operates was there massive cell loss accompanied by reduction in area and change in shape of the nucleus. These changes were most obvious in the caudal and ventrolateral portions of the C-MPRN. Pooled data from each group of operates indicated that significantly more cells were lost in the C-MPRN in the newborn than in the adult operates (p less than 0.01). Neurons of the C-MPRN which are known to project to the lower cervical and upper thoracic segments of the spinal cord (Brown, '74; Gwyn, '71) remained undamaged after the mid-thoracic hemisection in both groups. However, neurons of this region were enlarged in both groups when compared to a similar region of the ipsilateral MPRN. These neurons were found to be more enlarged in the newborn than in the adult operates (p less than 0.01). This result indicates that massive retrograde cell death takes place after a mid-thoracic hemisection in the neonatal rat. The retrograde degeneration of axotomized neurons partially may explain why CNS regeneration is not found in the immature mammal even though many of the factors thought to limit regeneration in the adult mammal may not be apparent. The increase in cell size of C-MPRN neurons which remain in the neonatal animals after mid-thoracic hemisection may be related to the increase in axonal size found in the region of the rubrospinal tract rostral to the thoracic lesion reported earlier (Prendergast and Stelzner, '76a). Both the increase in axonal and perikaryal size are hypothesized to be related to the increased distribution of supraspinal axons found in the gray matter rostral to a hemisection of the neonatal rat spinal cord.

Mid-thoracic spinal cord transection produces dramatically different behavioral results depending upon a rat's age at the time of surgery. The present study was initiated to determine whether the synaptic development in the gray matter of the normal, developing spinal cord differs before and after the period when maximal behavioral recovery occurs. The L6 segments from 10 groups of animals, 0--30 days of age, taken at 3 day intervals (4 animals/group) were studied by light microscopy. Areal measurements of the gray matter were made using an integrating x-y tablet interfaced to a computer. Cell size, cell density and area of neuropil were evaluated in the lateral portions of the intermediate gray matter, laminae VI and VII. Electron microscopic analyses of synaptogenesis were performed on material from the same region in animals 3, 12, 15, 21 and 30 days old using similar morphometric methods while taking note of vesicle, junctional, and mitochondrial morphology. A 60% increase in area of neuropil paralleled a linear increase, of comparable magnitude, in area of the gray matter until 15 days of age when both curves reached plateau. Neuronal perikaryal size remained constant (congruent to 200 sq. microns in plane of nucleolus) throughout development and so could not have contributed to the increase in area of gray matter. Areal measurements of the size and counts of the number of vesicle containing profiles demonstrated a 50% increase in density of axon terminals between 3 and 12 days of age and a steady decline thereafter. The size of vesicle-containing profiles in laminae VI and VII remained constant at a small value (congruent to 0.35 sq microns) until 12 days of age, showed rapid growth to 0.54 sq. microns between 12 and 15 days of age, followed by a more moderate increase in sectional area after 15 days. These results suggest that during the period when recovery of function follows spinal injury, synaptogenesis in the intermediate gray region of the lumbar spinal cord proceeds rapidly, while at stages when little recovery of function follows spinal transection, synaptogenesis is essentially complete.

Even though optic regeneration is successful in the frog, Rana pipiens, at completion considerable ganglion cell loss has occurred. To determine whether ganglion cell loss affects the size of the remaining ganglion cells, these cells were back-filled with horseradish peroxidase. The size of one class of ganglion cell 6 months to 1 year following nerve crush injury (N = 4) was compared to that of normal cells of this class (N = 4). The average area of the perikaryon was 35% larger than normal (less than 0.01). This change is interpreted to reflect the increased metabolic needs of the neuron required to maintain a larger than normal axonal arbor.

Retrograde axonal transport was used to determine which ascending nerve tracts from the lumbosacral spinal cord are present in the cervical spinal cord of the newborn rat and if their cell bodies survive axotomy. A pledget of true blue was applied to a low cervical spinal transection in the newborn rat (N = 4). After a 5-day survival period, neurons were labeled in the laminae of origin of all ascending nerve tracts throughout the lumbosacral spinal cord. Neurons labeled in the same way survived for at least 1 month postoperatively when the spinal cord was transected at a midthoracic level at 5 days of age (N = 4). No neurons in the lumbosacral spinal cord were labeled if the midthoracic spinal cord was transected at the same time as application of the dye to cervical spinal cord (N = 2). Therefore, neurons labeled with true blue from cervical spinal cord during the neonatal period are likely to have been axotomized by thoracic injury made at 5 days of age. Three months after midthoracic spinal transection of newborn rats, HRP was injected or a pledget was applied to the first spinal segment caudal to this lesion (N = 8). The same population of neurons was labeled as in adult rats receiving application of HRP to an acute midthoracic spinal transection (N = 4). Neurons were seldom labeled in adult rats in which HRP was injected and ascending nerve tract axons not damaged (N = 4). These results suggest that most ascending nerve tract axons are present in cervical spinal cord during the neonatal period (by 4 to 5 days of age).(ABSTRACT TRUNCATED AT 250 WORDS)

Albino rats, 0, 9, 12, 15, 18, 21 or greater than 90 days of age, were given a mid-thoracic spinal cord transection. Evaluation of responses of the hindlimbs to a variety of behavioral tasks was begun on the day of surgery and at intervals throughout the postoperative survival period (up to 300 days). Two investigators, independently and without knowledge of the animals' ages or survival times, rated the response data. Histological study showed all transections to be complete. Large differences in behavior are observed when animals trasected at the neonatal stage (0-4 days of age) are compared with animals transected at the weanling stage (21-26 days of age)37. Results of the present investigation indicate a critical period near 15 days of age; animals lesioned prior to this age (0, 9, 12 days of age) show response development and recovery similar to the neonatally lesioned animal, whereas those animals lesioned at a later age (18, 21, greater than 90 days of age) show little recovery and are behaviorally similar to the weanling transected animal. In animals lesioned prior to the fifteenth postnatal day, postural responses appear depressed for a brief period but recover rapidly while most responses of animals in the older groups are depressed for longer periods and never attain the degree of recovery characteristic of the neonatally transected animal. Finally, like the neonatally transected animal, rats lesioned on the ninth and twelfth postnatal day develop certain responses at appropriate times relative to normal response development. If, however, these responses are mature and supraspinal control is present at the time of lesioning, they appear to be permanently depressed and fail to recover.

The spinal cords of newborn and weanling rats were hemisected at the mid-thoracic level. Control studies revealed that Fink-Heimer positive debris was absent in the gray matter at three months postoperative. The remaining animals were given a second lesion, a high cervical spinal hemisection, at five to seven months after the original thoracic hemisection. The pattern of degeneration rostal to the thoracic lesion was compared with similar regions of the spinal cord from animals receiving only a cervical hemisection at the adult stage. In neither experimental group of doubly hemisected rats was there any degeneration observed below the thoracic lesion site, even though no glial or connective tissue scar had formed in animals originally operated at birth. Thus no regeneration had occurred. At least one segment above the initial hemisection: 1. the majority of degenerating axons were localized toward the lateral edge of the spinal cord, especially in the doubly lesioned neonatal group; 2. the erae of ipsilateral white matter was reduced more in the neonatal than the weanling operates; 3. there was an upward shift in axonal diameter of ipsilateral fibers in both the region of the rubrospinal tract and the ventrolateral portion of the lateral funiculus of the doubly hemisected rats when compared with the cervically lesioned controls; 4. a significantly greater amount of degeneration was present in lamina VII of Rexed in both the neonatal and weanling experimental operates (p less than 0.05 weanling; p less than 0.001 neonate); 5. no mean difference in area was seen between the ipsilateral and contralateral gray matter in any group for the segments of the spinal cord in which the judgements and measurements were taken. These data suggest that there has been sprouting of axons from descending nerve tracts rostral to the thoracic lesion in both the neonatal and weanling experimental groups. The question remains whether the sprouting of descending nerve tracts is from collateral of axons which normally project rostral to the thoracic hemisection and are not cut by the thoracic lesion (collateral sprouting) or from collaterals of lesioned axons (regenerative sprouting). Present evidence favors collateral sprouting, expecially in the neonatal operate where much retrograde cell death appears to have taken place.

The maturation of lamina IX neurons in the thoracic spinal cord of the rat was studied from day 13 of gestation until adulthood using a modified Golgi-Cox stain. The large multipolar neurons developed in an orderly and progressive manner. At day G-13, the round or stellate perikarya were smooth. Primary and secondary dendrites were adult in number with numerous irregular expansions and growth cones. Tertiary dendrites were beginning to form. Between day G-18 and postnatal day 5 the irregular surface of the perikarya and all portions of the dendrites had numerous long spines and filopodia. Growth cones were present at the end of terminal dendrites and also along the shafts of secondary and tertiary dendrites. By postnatal day 11 there was a decrease in the number of spines on the perikarya and proximal dendrites but spines remained prominent on secondary and tertiary dendrites. Between postnatal days 18 and 31 the adult morphology was attained. The perikarya and dendrites of adult neurons were devoid of spines except for occasional spines on the distal half of the dendritic tree. Elongated smooth varcosities were present except for the distal half of terminal dendrites where these expansions were more beaded. Although these morphologic changes were, in certain respects, the reciprocal of the regressive changes seen in these neurons after spinal hemisection in the adult rat (3), a major difference was the apparent absence of a "spiny stage" in the denervated lamina IX neurons of the adult.

Previous experiments have shown that a substantial number of regenerating optic axons in adult frogs (Rana pipiens) are misrouted into the opposite optic nerve and retina during early stages of regeneration. This projection is maximal at 5 and 6 weeks after optic nerve crush. To further characterize this anomalous projection, small quantities of horseradish peroxidase (HRP) were injected into the right eye or right optic nerve 5 or 6 weeks after right optic nerve crush. Twenty-four hours later the animals were killed and regenerating axons anterogradely filled with HRP were reacted with the tetramethyl-benzidine method or a diaminobenzidine-CoCl2 method. Serial reconstruction tracing the course of individual axons through the optic chiasm showed that few of the axons projecting into the opposite optic nerve were collaterals of axons projecting centrally. Instead, the majority of labeled axons misdirected into the opposite nerve or contributing to an expanded projection into the ipsilateral optic tract turned out of the chiasm without branching. Many of the labeled regenerating axons had unusual trajectories within the chiasm, making abrupt turns or changing their direction of growth. Most of the axons misrouted into the opposite nerve came from portions of the chiasm nearest to the nerve of other eye. In three of eight frogs with an intact optic nerve, a small number of HRP-labeled axons were found in the left nerve after right nerve injection, but there was no indication that these axons reached the left eye. The results from this investigation suggest that the most parsimonious explanation for the chiasmal misrouting of regenerating frog optic axons is that axons are mechanically deflected into inappropriate pathways.