Unoperated Control Animals Research Articles

Hippocampal lesions impair non-navigational spatial memory in macaques.

Decades of studies robustly support a critical role for the hippocampus in spatial memory across a wide range of species. Hippocampal damage produces clear and consistent deficits in allocentric spatial memory that requires navigating through space in rodents, non-human primates, and humans. By contrast, damage to the hippocampus spares performance in most non-navigational spatial memory tasks-which can typically be resolved using egocentric cues. We previously found that transient inactivation of the hippocampus impairs performance in the Hamilton Search Task (HST), a self-ordered non-navigational spatial search task. A key question, however, still needs to be addressed. Acute, reversible inactivation of the hippocampus may have resulted in an impairment in the HST because this approach does not allow for neuroplastic compensation, may prevent the development of an alternative learning strategy, and/or may produce network-based effects that disrupt performance. We compared learning and performance on the HST in male rhesus macaques (six unoperated control animals and six animals that underwent excitotoxic lesions of the hippocampus). We found a significant impairment in animals with hippocampal lesions. While control animals improved in performance over the course of 45 days of training, performance in animals with hippocampal lesions remained at chance levels. The HST thus represents a sensitive assay for probing the integrity of the hippocampus in non-human primates. These data provide evidence demonstrating that the hippocampus is critical for this type of non-navigational spatial memory, and help to reconcile the many null findings previously reported.

MP07-03 ASSESSMENT OF BLADDER SENSATION AFTER YEAR-LONG LOWER SPINAL ROOT TRANSECTION AND SURGICAL REINNERVATION USING BEHAVIORAL MEASURES

INTRODUCTION AND OBJECTIVES:We aim to surgically reinnervate a lower motor neuron neurogenic bladder via obturator-to-pelvic and sciatic-to-pudendal nerve transfer. Behavioral observations provide the most global assessment of functional recovery. We sought to determine whether observed micturition postures disappear in decentralized animals and reappear after reinnervation.METHODS:Three groups of female canines were used: 1) Long-term survival (12 months) after transection of all ventral and dorsal sacral roots, the dorsal roots of L7 and hypogastric nerves, bilaterally (n=4); 2) Procedures described in Group 1, followed by obturator-to-pelvic nerve and sciatic-to-pudendal nerve transfer (n=3); 3) Unoperated control animals (n=3). Micturition postures were monitored for 24 hours at monthly intervals via video recording. Bacteriuria was tracked midway through the study with urine Multistix test strip assays, body temperature, and cultured urine specimens by a commercial clinical laboratory. Awake cystomet...

Bacterial Dissemination to the Brain in Sepsis.

Sepsis causes brain dysfunction and neuroinflammation. It is unknown whether neuroinflammation in sepsis is initiated by dissemination of bacteria to the brain and sustained by persistent infection, or whether neuroinflammation is a sterile process resulting solely from circulating inflammatory mediators. To determine if gut bacteria translocate to the brain during sepsis, and are associated with neuroinflammation. Murine sepsis was induced using cecal ligation and puncture, and sepsis survivor mice were compared with sham and unoperated control animals. Brain tissue of patients who died of sepsis was compared with patients who died of noninfectious causes. Bacterial taxa were characterized by 16S ribosomal RNA gene sequencing in both murine and human brain specimens; compared among sepsis and nonsepsis groups; and correlated with levels of S100A8, a marker of neuroinflammation using permutational multivariate ANOVA. Viable gut-associated bacteria were enriched in the brains of mice 5 days after surviving abdominal sepsis (P < 0.01), and undetectable by 14 days. The community structure of brain-associated bacteria correlated with severity of neuroinflammation (P < 0.001). Furthermore, bacterial taxa detected in brains of humans who die of sepsis were distinct from those who died of noninfectious causes (P < 0.001) and correlated with S100A8/A9 expression (P < 0.05). Although bacterial translocation is associated with acute neuroinflammation in murine sepsis, bacterial translocation did not result in chronic cerebral infection. Postmortem analysis of patients who die of sepsis suggests a role for bacteria in acute brain dysfunction in sepsis. Further work is needed to determine if modifying gut-associated bacterial communities modulates brain dysfunction after sepsis.

Experimental Control for the Ovariectomized Rat Model: Use of Sham Versus Nonmanipulated Animal

One of 2 models required by the U.S. Food and Drug Administration for registration of a treatment for osteoporosis, the ovariectomized (OVX) rat model, is widely used in scientific studies investigating sex hormone-deficient bone loss. The use of control nonhuman animals is critical because bone turnover may be affected by animal stress, use of anesthetic, and the mechanisms involved in wound healing. Historically, researchers have used sham-operated animals who undergo the same manipulations as the OVX rats, but ethical concerns require consideration of unmanipulated (unoperated) control animals to minimize animal distress and unnecessary procedures. Herein, we report the results of 3 studies including OVX, sham, and unmanipulated rats and the effects on bone mineral density and content (BMD/BMC) during 2 to 6 months postsurgery. Our data indicate that while OVX animals generally had lower BMD and BMC than animals in either of the control groups, no differences were observed between sham and unmanipulated animals at any of the time points assessed. However, because bone turnover is a long-term process, studies with longer duration and multiple endpoints are warranted to confirm these results.

Chronic compression of mouse dorsal root ganglion alters voltage-gated sodium and potassium currents in medium-sized dorsal root ganglion neurons

Chronic compression (CCD) of the dorsal root ganglion (DRG) is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. Previously, we examined electrophysiological changes in small-diameter lumbar level 3 (L3) and L4 DRG neurons treated with CCD; the present study extends these observations to medium-sized DRG neurons, which mediate additional sensory modalities, both nociceptive and non-nociceptive. Whole-cell patch-clamp recordings were obtained from medium-sized somata in the intact DRG in vitro. Compared with neurons from unoperated control animals, CCD neurons exhibited a decrease in the current threshold for action potential generation. In the CCD group, current densities of TTX-resistant and TTX-sensitive Na(+) current were increased, whereas the density of delayed rectifier voltage-dependent K(+) current was decreased. No change was observed in the transient or "A" current after CCD. We conclude that CCD in the mouse produces hyperexcitability in medium-sized DRG neurons, and the hyperexcitability is associated with an increased density of Na(+) current and a decreased density of delayed rectifier voltage-dependent K(+) current.

Selective changes in foraging behavior following bilateral neurotoxic amygdala lesions in rhesus monkeys.

Across a variety of species, the amygdala appears to play a key role in the detection and avoidance of potential dangers (e.g., unfamiliar social partners, novel objects or contexts, potential predators, etc.). For many species, seeking out appropriate food sources and avoiding novel, distasteful or potentially tainted food is also a daily concern. Amygdala damage in nonhuman primates has been linked to increased willingness to select unfamiliar or unpalatable foods, as well as inedible items that intact animals typically reject. However, such findings have not always been consistent and have typically been observed in relatively restrictive, laboratory-based testing contexts. We evaluated the food choices of six adult male rhesus monkeys (Macaca mulatta) with bilateral, neurotoxic amygdala lesions and six age- and experienced-matched unoperated control animals. Each animal was able to forage freely in a large enclosure stocked with five preferred and five nonpreferred foods that changed locations each day. While both groups quickly selected palatable foods, monkeys with amygdala lesions consistently selected unpalatable foods that the unoperated control animals generally avoided. Even after repeated presentations of the unpalatable foods, the amygdala-lesioned monkeys failed to change their initial pattern of diminished avoidance. These results are consistent with a general role for the amygdala in danger detection and prevention of harm in the presence of novel or noxious stimuli, regardless of whether such stimuli are conspecifics, predators, objects or foods.

Effects Of Pınealectomy On Morphologıcal Features Of Blood Vessel In Chıcken: An Electron Mıcroscopıc And Stereologıcal Study

Although the effects of melatonin on vascular smooth muscle and vascular reactivity are described in last decade, the effects of melatonin on vascular tissues are still vague. The current study investigated the effects of pinealectomy on the blood vessel wall in the chicken. Fifteen chicks were divided into three groups, namely unoperated control, sham-operated and pinealectomized chicks. At the end of the experiment, a quantitative structural analysis of 15 sciatic artery samples from these groups was investigated. Effects of pinealectomy were quantified using volume fraction approach of stereological method. In this study, total cross-sectional area of the artery in pinealectomy group was significantly higher than those in unoperated control and sham-operated animals (p < 0.05). However, surgical pinealectomy procedure resulted in a significantly decreased thickness of the tunica adventitia of the vessel wall, in contrast to those of tunica media (p < 0.01). The present results indicate that pinealectomy had a vasodilator effect on the sciatic artery. In the light of current study, the structural changes in the vessel demonstrated by quantitative morphometric methods have been interpreted as a reflection of the role of melatonin on vessel wall and vascular reactivity, but this suggestion need to be validated in the human setting.

Hippocampal Lesions in Rhesus Monkeys Disrupt Emotional Responses but Not Reinforcer Devaluation Effects

Although the role of the hippocampus in emotional behavior has long been recognized, the extent to which the hippocampus plays a role in the regulation and expression of emotion in rhesus monkeys has not been systematically explored. Rhesus monkeys (Macaca mulatta) with excitotoxic lesions of the hippocampal formation and unoperated control animals were assessed on two different types of emotional processing: defensive reactions to a potential predator (experiment 1) and ability to update the value of positive reinforcers, in this case food (experiment 2). Monkeys with aspiration lesions of the perirhinal cortex were also included in this study as an operated control group. In experiment 1, whereas both operated groups showed reduced latencies to retrieve food located near an innately fear-provoking stimulus, a fake snake, only monkeys with hippocampal lesions displayed reduced defensive reactions to the snake. In experiment 2, both operated groups performed as well as control animals when choosing objects flexibly based on the current value of a food. These findings dissociate the hippocampus and perirhinal cortex in fear expression and specifically implicate the hippocampal formation in generating responses to stimuli that are potentially threatening.

Sensitization of pain-modulating neurons in the rostral ventromedial medulla after peripheral nerve injury.

Nerve injury can lead to mechanical hypersensitivity in both humans and animal models, such that innocuous touch produces pain. Recent functional studies have demonstrated a critical role for descending pain-facilitating influences from the rostral ventromedial medulla (RVM) in neuropathic pain, but the underlying mechanisms and properties of the relevant neurons within the RVM are essentially unknown. We therefore characterized mechanical responsiveness of physiologically characterized neurons in the RVM after spinal nerve ligation, a model of neuropathic pain that produces robust mechanical hyperalgesia and allodynia. RVM neurons were studied 7-14 d after spinal nerve ligation, and classified as "on-cells," "off-cells," or "neutral cells" using standard criteria of changes in firing associated with heat-evoked reflexes. On-cells are known to promote nociception, and off-cells to suppress nociception, whereas the role of neutral cells in pain modulation remains an open question. Neuronal and behavioral responses to innocuous and noxious mechanical stimulation were tested using calibrated von Frey filaments (4-100 g) applied to the hindpaws ipsilateral and contralateral to the injury, and in sham-operated and unoperated control animals. On- and off-cells recorded in nerve-injured animals exhibited novel responses to innocuous mechanical stimulation, and enhanced responses to noxious mechanical stimulation. Neuronal hypersensitivity in the RVM was correlated with behavioral hypersensitivity. Neutral cells remained unresponsive to cutaneous stimulation after nerve injury. These data demonstrate that both on- and off-cells in the RVM are sensitized to innocuous and noxious mechanical stimuli after nerve injury. This sensitization likely contributes to allodynia and hyperalgesia of neuropathic pain states.

INTRAHEPATIC STAT-3 DNA BINDING DOES NOT CORRELATE WITH INTRANUCLEAR PHOSPHO-STAT-3 LEVELS IN SEPTIC MICE

Objective: Intrahepatic signal transducer and activator of transcription (STAT)-3 activation and acute phase gene expression of α2-macroglobulin (A2M) has shown to predict outcome after cecal ligation and double puncture (2CLP) in rats. Previous studies have shown that STAT-3 DNA binding is decreased dramatically in mice 16 and 24 hours following 2CLP and remains attenuated compared to single puncture (CLP). We hypothesize that this decrease is from a disruption of the intracellular STAT-3 signaling pathway. Methods: Liver homogenates were collected from 6-8 weeks old C57 B1/6 mice 3, 6, 16, 24, 48, and 72 hours after sham operation (SO) and 2CLP. Liver homogenates from unoperated control (T0) animals were also collected. Nuclear and cytosolic protein extracts were made from liver homogenates. Electrophoretic mobility shift assays (EMSA) were previously done on nuclear extracts detailing STAT-3 DNA binding activity following SO, and 2CLP. Western blotting was performed on hepatic nuclear protein extracts following SO and 2CLP for phosphorylated- STAT-3 (p-STAT-3). Results: Hepatic intranuclear p-STAT-3 levels were undetectable in T0 animals. However, significant levels were detected in samples taken 3 and 6 hours after 2CLP. Intranuclear p-STAT-3 levels were decreased at 16 hours and undetectable at 24 hours post 2CLP. However, levels increased again 48 and 72 hours post 2CLP. A similar pattern but markedly decreased intranuclear p-STAT-3 levels were observed after SO and there were no detectable levels 48 and 72 hours post SO. Conclusion: Intranuclear p-STAT-3 levels initially correlated with STAT-3 DNA binding, but did not last beyond 24 hours in 2CLP. Although there is signal attenuation 16 and 24 hours after 2CLP, the lack of STAT-3 DNA binding despite elevated intranuclear p-STAT-3 levels 48 and 72 hours post 2CLP suggests a defect with intranuclear STAT-3 regulation.

Myocardial infarction stimulates galanin expression in cardiac sympathetic neurons

Cardiac ischemia-reperfusion alters sympathetic neurotransmission in the heart, but little is known about its effect on neuropeptide expression in sympathetic neurons. Ischemia followed by reperfusion induces the production of inflammatory cytokines in the heart, including interleukin-6 and cardiotrophin-1. These cytokines and related molecules inhibit the expression of neuropeptide Y (NPY), and stimulate the expression of vasoactive intestinal peptide (VIP), substance P (SubP), and galanin (GAL) in cultured sympathetic neurons. Therefore, we quantified NPY, VIP, SubP, and GAL mRNA in neurons of the stellate ganglia 1 week after ischemia-reperfusion to determine if neuropeptide expression was altered in cardiac sympathetic neurons. NPY, VIP, and SubP mRNAs were unchanged compared to unoperated control animals, but GAL mRNA was increased significantly. The increased GAL mRNA was not accompanied by elevated GAL peptide content in the stellate ganglia. Galanin content was increased significantly in the heart, however, indicating that elevated GAL mRNA led to increased peptide production. GAL content was increased in the left ventricle below the coronary artery ligation, but was not increased significantly in the atria or the base of the heart above the ligation. The buildup of GAL specifically in the damaged left ventricle is consistent with previous reports that GAL is transported to regenerating nerve endings after axon damage.

Failure to form a stable topographic map during optic nerve regeneration: abnormal activity-dependent mechanisms

Visually evoked responses in the optic tectum are mediated by glutamate receptors. During development, there is a switch from N-methyl- d-aspartate (NMDA)- to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated activity as the retinotectal map refines and visual function ensues. A similar pattern is seen in goldfish as the map refines during optic nerve regeneration. Here we examined glutamate receptors during optic nerve regeneration in the lizard, Ctenophorus ornatus, in which an imprecise retinotopic map forms transiently but degrades, leaving animals blind via the experimental eye. Receptor function was examined using NMDA and AMPA/kainate antagonists during in vitro tectal recording of visually evoked post-synaptic extracellular responses. Expression of NR1 (NMDA) and GluR2 (AMPA) receptor subtypes was examined immunohistochemically. In unoperated control animals, responses were robust and AMPA/kainate receptor-mediated. When the imprecise map was present, responses were difficult to evoke and insecure; periods of spontaneous activity as well as inactivity were also noted. Although AMPA/kainate-mediated activity persisted and GluR2 immunoreactivity increased transiently, NMDA receptor-mediated activity was also consistently detected and NR1 expression increased. In the long term, when the map had degraded, responses were readily evoked and predominantly AMPA/kainate receptor-mediated although some NMDA-mediated activity and NR1 expression remained. We suggest that the asynchronous activity reaching the optic tectum results in an inability to recapitulate the appropriate functional sequences of expression of NMDA and AMPA/kainate receptors necessary to refine the retinotectal map.

Myocardial apoptosis after cardioplegic arrest in the neonatal lamb

Objective Myocardial apoptosis is observed after various cardiac injuries and is also a normal part of fetal cardiac development and early postnatal maturation. Cardioplegic arrest and reperfusion result in ischemic injury and oxidative stress, known triggers of apoptosis. Because the neonatal heart is in a proapoptotic state, we hypothesize that apoptosis is triggered after cardioplegic arrest in neonatal myocardium. Methods We started neonatal lambs (6-8 days old, n = 5) on cardiopulmonary bypass and administered cold crystalloid cardioplegia at 20-minute intervals. Total crossclamp time was 70 minutes, and bypass time was 90 minutes. After a six-hour recovery period, the hearts were excised and examined by using TdT-mediated dUTP nick-end labeling; radiolabeled DNA electrophoresis; fluorimetric caspase 3, 8, and 9 activity assay; mRNA microarray; and Western immunoblotting. Control lambs were anesthetized but did not undergo operation (n = 5) or were started on cardiopulmonary bypass for 90 minutes but not arrested (n = 5). Results Lambs subjected to cardioplegia had 5-fold more TdT-mediated dUTP nick-end labeling–positive nuclei compared with that seen in unoperated control animals ( P = .007) and bypass-only control animals ( P = .008). DNA laddering was present in all postcardioplegia hearts but absent among control hearts. Bad and Bcl-X mRNA transcription increased significantly. Caspase 3, 8, and 9 activities were slightly greater than those seen in control animals, but the differences were not significant. No change was detected in Bcl-2, Bax, or Bcl-xL proteins. Conclusions In a clinically relevant model of neonatal cardioplegic arrest, increased apoptotic cell death is present 6 hours after reperfusion, and both proapoptotic and antiapoptotic responses are triggered. The clinical implications of apoptosis after cardioplegic arrest remain undetermined.

A Role for Platelet-Derived Growth Factor-BB in Rat Postpneumonectomy Compensatory Lung Growth

Unilateral pneumonectomy leads to compensatory growth in the residual lung, the mediators of which are largely unknown. We hypothesized, based on its other known roles in lung cell growth, that platelet-derived growth factor (PDGF)-BB would be an essential mediator of postpneumonectomy compensatory lung growth. Left-sided pneumonectomies were performed on 21-d-old rats, for comparison with sham-operated or unoperated control animals. Body weights were not different between groups. Right lung weights and DNA content were significantly increased (p < 0.05), compared with controls, by 10 d after pneumonectomy. The rate of DNA synthesis was maximal on d 5 postpneumonectomy. Total right lung PDGF-B mRNA and PDGF-BB protein increased after pneumonectomy, but were apparently tightly regulated, relative to total right lung beta-actin mRNA and protein content, respectively. However, PDGF-BB expression after pneumonectomy was apparently not purely constitutive, in that daily i.p. injections of a truncated soluble PDGF beta-receptor both reduced activation of the native PDGF beta-receptor, and attenuated increased lung DNA synthesis on d 3 after pneumonectomy. These findings are consistent with a critical role for PDGF-BB in postpneumonectomy lung growth.

Effect of the distal remnant on ileal adaptation

The ileum has a greater adaptive capacity than the jejunum after intestinal resection, which may be, in part, related to increased exposure to luminal contents and intrinsic properties of the ileum. However, the intestinal remnant might contribute to this adaptive response as well. Our aim was to determine the effect of the distal intestinal remnant on ileal adaptation when the ileum is proximal in the intestinal tract. Twenty-one Lewis rats were included in the study. One group (n = 7) served as unoperated control subjects, the second group (n = 7) underwent transposition of the jejunum and ileum, and the third group (n = 7) underwent 50% proximal resection with syngeneic transplantation of the ileum. Nutritional status and structural adaptation were studied at 14 days. Animals in both the transposition and transplant groups initially lost weight but weights returned to above preoperative levels at 14 days. Food intake, stool weight, and serum albumin levels were similar in these two groups. Intestinal weight and diameter were similar in the proximal end of the ileal segment in the two study groups and were significantly increased compared to control values (0.26 ± 0.04 and 0.31 ± 0.02 vs. 0.10 ± 0.0 g/cm and 8.4 ± 0.5 and 9.1 ± 0.8 vs. 4.9 ± 0.3 mm; P <0.05). Intestinal weight and diameter of the distal end of the ileal segment were greater than those values in unoperated control animals but were greatest in the ileal transplant group (0.15 ± 0.1 and 0.24 ± 0.03 vs. 0.07 ± 0.01 g/cm and 5.6 ± 1.1 and 8.7 ± 0.6 vs. 4.3 ± 0.2 mm; P <0.05). Villus height and crypt depth were similar in both the proximal and distal ends of the ileal segments in the two study groups and were significantly increased compared to control values (642 ± 75 and 720 ± 15 vs. 411 ± 24 proximal and 443 ± 49 and 500 ± 46 vs. 343 ± 22 μm distal, P <0.05; 223 ± 34 and 244 ± 33 vs. 173 ± 20 proximal and 192 ± 28 and 209 ± 18 vs. 144 ± 26 μm distal, P <0.05). Proximal placement of the ileum by either transposition or transplantation results in structural adaptation. This occurs to a similar extent whether the distal remnant is jejunum or ileum. Thus increased exposure to luminal contents and intrinsic properties appear to be the important factors in the adaptive capability of the ileum when the ileum is the proximal portion of the intestinal tract.

Regulation of ?7 nicotinic acetylcholine receptors in mouse somatosensory cortex following whisker removal at birth

Previous studies in postnatal mouse demonstrating high levels of alpha7 nicotinic acetylcholine receptors on layer IV somatosensory cortical neurons coincident with the onset of functional synaptic transmission led us to investigate whether the number and/or the localization of these receptors could be regulated by activity. Accordingly, we examined alpha-bungarotoxin binding in mouse somatosensory cortex following removal of all of the vibrissae on one side of the face, either by vibrissal follicle cauterization or daily plucking beginning on the day of birth. Following vibrissa plucking, the levels of [125I]alpha-bungarotoxin binding on postnatal day 6 were significantly higher (23 +/- 7%) in the denervated cortex (contralateral to the peripheral manipulation) than the intact cortex. Cauterization also resulted in significantly higher (14 +/- 3%) [125I]alpha-bungarotoxin binding in the contralateral vs. the ipsilateral cortex. In contrast, there was no difference in [125I]alpha-bungarotoxin binding in the left and right cortices of unoperated control animals. At postnatal day 14, levels of [125I]alpha-bungarotoxin binding in layer IV were very low in control animals as well as in animals subjected to whisker plucking or cautery. These findings suggest that reducing activity in the somatosensory pathway regulates the density of alpha7 nicotinic acetylcholine receptors during the first postnatal week. However, the normal decrease in receptor density that is seen during the second postnatal week of development proceeds despite altered sensory activity.

The effects of cytotoxic entorhinal lesions and electrolytic medial septal lesions on the acquisition and retention of a spatial working memory task.

Rats with lesions either of medial septal nucleus (MSN) or the entorhinal cortex (ECx) were compared postoperatively with unoperated controls in a discrete-trial, delayed matching-to-position (DMTP) task, conducted on an elevated T-maze. A DMTP trial consisted of two consecutive visits to the maze: an information run and a choice run. The animals were first forced to visit a randomly selected choice arm in the information run. In the choice run, the correct response was to match the choice arm that had been visited on the information run, regardless of whether the information run itself had been rewarded or not. MSN animals failed to succeed in this task, performing at close to chance level throughout training. On the other hand, ECx rats consistently perform at a level comparable with that of unoperated controls; both groups attained more than 90% correct after 192 trials. Long-term retention testing was carried out after an intermission of 4 weeks, when the same task was re-administered to the ECx and unoperated control animals. ECx animals showed significantly less saving than controls in the retention test. In contrast, when the retention interval within a DMTP trial was increased by the imposition of a 20-s delay between the information and choice runs, the ECx group was not selectively affected by this manipulation.

Neutralizing antibody to TGFβ modulates stromal fibrosis but not regression of photoablative effect following PRK

Previous studies have suggested that corneal fibrosis controlled by a TGFbeta-mediated cytocrine pathway underlies the development of clinical corneal haze and associated regression of photoablative effect following excimer laser PRK. Using a unique blocking antibody, we evaluated the role of TGFbeta in post-PRK corneal wound healing as measured by in vivo Confocal Microscopy Through Focusing (CMTF). Twelve rabbits received a monocular, 6-mm diameter, 9.0 D PRK myopic correction. Six animals received 50 microg of anti-TGFbeta blocking antibodies applied topically 3x/day for three days post-PRK, while six animals received vehicle alone. An additional six animals served as unoperated controls. At various times during a four-month-period, animals were evaluated using CMTF, which generates a quantitative image intensity depth profile of the cornea. The location and reflectivity of corneal structures were identified from CMTF-profiles and used to determine epithelial and stromal thickness and corneal light reflectivity as an objective estimate of corneal haze. To correlate in vivo and ex vivo morphology, an additional six rabbits were analyzed at differing temporal intervals post-PRK for the expression and cytoskeletal organization of contractile microfilaments: f-actin (stress fibers) and alpha-smooth muscle actin (a molecular marker for myofibroblast transformation). Anti-TGFbeta treated corneas showed significantly less CMTF measured light reflectivity (ANOVA, p < 0.02) following PRK compared to vehicle treated corneas with a 34% decrease at two weeks (2513 +/- 758 U compared to 3810 +/- 1262 U) and a 61% reduction in reflectivity at four months (447 +/- 208 U compared to 1154 +/- 585 U). The reduction in early development of light reflecting structures and the more rapid decline appeared related to anti-TGFbeta-mediated inhibition of keratocyte activation and proliferation, myofibroblast transformation, and stromal fibrosis. Between anti-TGFbeta and vehicle treated corneas, no significant differences were detected in either photoablation depth (126 +/- 9 microm versus 126 +/- 7 microm) or regression of photoablative effect (postoperative stromal thickening at four months: 95 +/- 16 microm versus 95 +/- 10 microm). Histologic examination demonstrated that regression of photoablative effect in anti-TGFbeta treated corneas was related entirely to regeneration by corneal growth underlying the photoablated stromal surface. In vehicle treated corneas, fibrosis or deposition of new fibrotic tissue above the photoablated stromal surface was observed but contributed only about 25% of the total postoperative stromal thickening. No epithelial hyperplasia was detected. In unoperated control animals, a physiologic stromal thickening of 5 +/- 2 microm per month (p < 0.001) was observed. This study confirms our earlier observations that increased corneal light reflectivity following PRK is predominantly due to: (1) distortion of the photoablated stromal surface leading to prominent reflections; and (2) increased reflections from activated and transformed keratocytes. Anti-TGFbeta reduced keratocyte activation and transformation and inhibited stromal fibrosis, leading to a reduction in early light reflectivity as well as to a more rapid decline. Of greatest interest is the unexpected finding that anti-TGFbeta treatment inhibited stromal fibrosis without reducing or delaying post-PRK stromal re-thickening. Based on these findings we propose that corneal thickness may be tightly and dynamically regulated by an unknown, non-TGFbeta mediated pathway. We propose that anti-TGFbeta treatment may be useful in reducing post-PRK corneal haze development in patients by: (1) inhibiting the recruitment of highly reflective, activated keratocytes, (2) inhibiting myofibroblast transformation, and 3) reducing stromal fibrosis.

Neutralizing antibody to TGFβ modulates stromal fibrosis but not regression of photoablative effect following PRK

Purpose. Previous studies have suggested that corneal fibrosis controlled by a TGFβ-mediated cytocrine pathway underlies the development of clinical corneal haze and associated regression of photoablative effect following excimer laser PRK. Using a unique blocking antibody, we evaluated the role of TGFβ in post-PRK corneal wound healing as measured by in vivo Con-focal Microscopy Through Focusing (CMTF).Methods. Twelve rabbits received a monocular, 6-mm diameter, 9.0 D PRK myopic correction. Six animals received 50 μg of anti-TGFβ blocking antibodies applied topically 3×/day for three days post-PRK, while six animals received vehicle alone. An additional six animals served as unoperated controls. At various times during a four-month-period, animals were evaluated using CMTF, which generates a quantitative image intensity depth profile of the cornea. The location and reflectivity of corneal structures were identified from CMTF-profiles and used to determine epithelial and stromal thickness and corneal light reflectivity as an objective estimate of corneal haze. To correlate in vivo and ex vivo morphology, an additional six rabbits were analyzed at differing temporal intervals post-PRK for the expression and cytoskeletal organization of contractile micro-filaments: f-actin (stress fibers) and α-smooth muscle actin (a molecular marker for myofibroblast transformation).Results. Anti-TGFβ treated corneas showed significantly less CMTF measured light reflectivity (ANOVA, p < 0.02) following PRK compared to vehicle treated corneas with a 34% decrease at two weeks (2513 ± 758 U compared to 3810 ± 1262 U) and a 61% reduction in reflectivity at four months (447 ± 208 U compared to 1154 ± 585 U). The reduction in early development of light reflecting structures and the more rapid decline appeared related to anti-TGFβ-mediated inhibition of keratocyte activation and proliferation, myofibroblast transformation, and stromal fibrosis. Between anti-TGFβ and vehicle treated corneas, no significant differences were detected in either photoablation depth (126 ± 9 μm versus 126 ± 7 μm) or regression of photoablative effect (postoperative stromal thickening at four months: 95 ± 16 μm versus 95 ± 10 μm). Histologic examination demonstrated that regression of photoablative effect in anti-TGFβ treated corneas was related entirely to regeneration by corneal growth underlying the photoablated stromal surface. In vehicle treated corneas, fibrosis or deposition of new fibrotic tissue above the photoablated stromal surface was observed but contributed only about 25% of the total postoperative stromal thickening. No epithelial hyperplasia was detected. In unoperated control animals, a physiologic stromal thickening of 5 ± 2 μm per month (p > 0.001) was observed.Conclusions. This study confirms our earlier observations that increased corneal light reflectivity following PRK is predominantly due to: (1) distortion of the photoablated stromal surface leading to prominent reflections; and (2) increased reflections from activated and transformed keratocytes. Anti-TGFβ reduced keratocyte activation and transformation and inhibited stromal fibrosis, leading to a reduction in early light reflectivity as well as to a more rapid decline. Of greatest interest is the unexpected finding that anti-TGFβ treatment inhibited stromal fibrosis without reducing or delaying post-PRK stromal re-thickening. Based on these findings we propose that corneal thickness may be tightly and dynamically regulated by an unknown, non-TGFβ mediated pathway. We propose that anti-TGFβ treatment may be useful in reducing post-PRK corneal haze development in patients by: (1) inhibiting the recruitment of highly reflective, activated keratocytes, (2) inhibiting myofibroblast transformation, and 3) reducing stromal fibrosis. Curr. Eye Res. 17:736–747, 1998.

Craniofacial growth after a period of unilateral masticatory function in young rabbits.

Changes in craniofacial growth after a period of unilateral masticatory function were studied in rabbits. 10-day-old animals were divided into 3 groups. In Group I, mandibular and maxillary molars were ground down 2x a week on the right side under general anesthesia until age 50 days, and were thereafter left to grow without grinding. In Group II, the right-side molars were ground until age 40 days on. Between days 40 and 60, grinding was performed on the left side. The animals were thereafter left to grow. Group III consisted of unoperated control animals. All of the animals were fed whole pellets and water ad libitum, and were sacrificed at age 100 days. There were measurable differences in growth after periods of unilateral masticatory function. The mandibular ramus was higher, the condylar processus was larger sagittally, and angles between the anterior or posterior borders of the condylar process and inferior border of mandible were smaller in the treated than in the control animals, and there were differences between right and left sides of the same animal in the maxilla and mandible. The inclination of the articular surface of the glenoid fossa was steeper anteriorly on both sides in the treated than in the controls. It was concluded that growth after a period of unilateral masticatory function in young rabbits does not rectify all of the asymmetric changes in the maxilla, mandible and glenoid fossa resulting from the asymmetric function.