[Effects of acute fear stress on spatial memory and neuronal plasticity in the medial prefrontal cortex in mice].

The purpose of this study was to investigate the effects of acute fear stress on the spatial memory and neuronal plasticity of medial prefrontal cortex (mPFC) neurons in mice, and to elucidate the mechanisms underlying mPFC plasticity and post-stress memory regulation. Male C57BL/6 mice (6 weeks old) were randomly divided into control group and stress group. Foot shock stress was applied to establish an acute fear stress model. Changes in spatial memory were examined by the Morris water maze test, and the dynamic changes in the spike encoding of pyramidal neurons and GABAergic neurons in the prelimbic cortex (PrL) and infralimbic cortex (IL) of mPFC were detected by whole-cell recording. The results showed that acute fear stress significantly enhanced the percentage of freezing and the number of freezing, reduced the average speed, decreased the escape latency during acquisition phase, extended the probing time in the first quadrant and shortened the probing time in the third quadrant during probe trial, increased inter-spike interval, energy barrier and absolute refractory period of GABAergic neurons in the PrL and pyramidal neurons in the IL, while decreased inter-spike interval, energy barrier and absolute refractory period of pyramidal neurons in the PrL and GABAergic neurons in the IL. These results suggest that acute fear stress can enhance the spatial memory of mice, elevate the excitability and function of the PrL, while deteriorate the excitability and function of the IL, and the underlying mechanism may involve the role of mPFC microcircuitry plasticity in spatial memory after stress.

Regulatory effects of introduction of an exogenous FGF2 gene on other growth factor genes in a healing tendon.

In this study of a tendon injury model, we investigated how injection of a vector incorporating one growth factor gene changes expression levels of multiple growth factor genes in the healing process. The flexor tendon of chicken toes was completely cut and repaired surgically. The tendons in the experimental arm were injected with an adeno-associated virus-2 vector incorporating basic fibroblast growth-factor gene, whereas the tendons in the control arm were not injected or injected with sham vectors. Using real-time polymerase chain reaction, we found that, within the tendon healing period, a set of growth factor genes-transforming growth factor-ß1, vascular endothelial growth factor, and connective tissue growth factor-were significantly up-regulated. Expression of the platelet-derived growth factor-B gene was not changed, and the insulin-like growth factor was down-regulated. A tendon marker gene, scleraxis, was significantly up-regulated in the period. Our study revealed an intriguing finding that introduction of one growth factor gene in the healing tendon modulated expression of multiple growth factor genes. We believe this study may have significant implications in determining the approach of gene therapy, and the findings substantiate that gene therapy using a single growth factor could affect multiple growth factors.

Molecular events of cellular apoptosis and proliferation in the early tendon healing period.

PURPOSE: Cellular proliferation is accompanied by cellular apoptosis. In the healing digital flexor tendon, molecular events concerning cellular apoptosis have not been investigated. This study aimed to investigate the relationship between cellular apoptosis and proliferation in early tendon healing. METHODS: The flexor digitorum profundus tendons of 50 long toes in 25 chickens were transected and were repaired surgically. On postoperative days 3, 7, 14, 21, and 28, we subjected tendons to in situ terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay to detect apoptotic cells, immunofluorescence staining with antibodies to proliferating cell nuclear antigen to assess proliferation, and Bcl-2, an anti-apoptotic protein, to assess responses suppressive to apoptosis. The positively labeled tenocytes were counted microscopically and compared statistically. We also stained sections with hematoxylin and eosin to observe their healing status. An additional 12 tendons (6 chickens) served as day 0 controls. RESULTS: Compared with tendons at day 0, the healing tendons had notably greater cellularity in both epitenon and endotenon areas. The total number of cells and number of TUNEL-positive cells peaked at day 3. At days 7 to 21, the number of proliferating cell nuclear antigen-positive cells peaked. At days 7 and 14, the cells positively stained with Bcl-2 peaked. At days 14 to 28, the total number of cells and TUNEL-positive cells decreased significantly compared with those at days 3 and 7, yet the numbers remained greater than those on day 0. CONCLUSIONS: Apoptosis in the healing tendons peaks at day 3, followed about 10 days later by the peak proliferation period. Because Bcl-2 serves to inhibit apoptosis, a later increase in Bcl-2-positive cells indicates that tendon apoptosis is inhibited. These findings indicate that tenocyte apoptosis is accelerated within several days after injury, followed by increases in cellular proliferation and activation of molecular events to inhibit apoptosis in 2 to 4 weeks.

Application of AAV2-mediated bFGF gene therapy on survival of ischemic flaps: effects of timing of gene transfer.

Necrosis of surgically transferred flaps is a major problem in reconstructive surgery. We investigated efficacy of a new vector system-adeno-associated viral 2 (AAV2)-mediated bFGF gene transfer to enhance survival of the ischemic flap. Thirty-eight Sprague-Dawley rats were divided into 3 gene therapy groups and 1 nontreated control of 9 or 10 each. 7.5 x 10(10) AAV2-bFGF viral particles were injected to the dorsum of each of the 29 rats; these rats were divided into 3 groups according to the timing of flap elevation. At the time of surgery, 1 week, and 2 weeks after surgery, flaps of 3 x 7 cm were raised. One week after surgery, flap viability was measured. Vascularization and immunohistochemical staining of the bFGF were evaluated of histologic sections. Flap viability was significantly improved by the AAV2-bFGF gene therapy at the time of surgery, and the flaps with the greatest survival area were found in the rats injected with AAV2-bFGF, 2 weeks before surgery. However, flap viability was significantly decreased by the gene therapy 1 week before surgery. Histologically, vascularity was increased in the groups with AAV2-bFGF injection and immunohistochemical staining showed greatly enhanced bFGF expression by gene transfer. The novel approach of AAV2-bFGF gene therapy shows encouraging manifestations in improving survival of flaps when the flaps are prefabricated during or 2 weeks before surgery.

Effectiveness of microRNA in Down-regulation of TGF-beta gene expression in digital flexor tendons of chickens: in vitro and in vivo study.

PURPOSE: Transforming growth factor (TGF)-beta is considered to be responsible for the formation of scars such as adhesions around healing digital flexor tendons. We proposed to deliver microRNAs (miRNAs) to silence expression of the TGF-beta1 gene and to investigate the effectiveness of miRNAs in down-regulation of the TGF-beta1 gene in vitro and in vivo. METHODS: We designed and engineered 4 miRNAs according to genetic sequences of chicken TGF-beta1. Four plasmid vectors harboring the respective engineered miRNAs and 1 control vector were constructed. We transfected 30 wells of cultured tenocytes with these vectors and harvested them 48 hours later. The gene expression levels were quantified using real-time polymerase chain reactions. Subsequently, the miRNA that most effectively silenced TGF-beta gene in vitro was tested on 25 chickens in vivo. The miRNA and control vectors were injected into the injured tendons, respectively. At 1 and 6 weeks after surgery, the tendons were analyzed for gene expression and protein production. RESULTS: In both in vitro and in vivo settings, delivery of miRNA to the tendon substantially down-regulated expression of the TGF-beta gene but did not affect expression of the collagen I gene. In the healing tendon, TGF-beta gene expression was significantly down-regulated by 50% to 60% at 1 and 6 weeks. At 6 weeks, the collagen III gene expression was significantly down-regulated by 55% at 6 weeks and the connective tissue growth factor gene was significantly down-regulated by 25%. At 6 weeks, TGF-beta protein was substantially decreased. CONCLUSIONS: MicroRNA significantly down-regulates expression of the TGF-beta in vitro and in vivo. Application of miRNA did not down-regulate expression of the collagen I, but downregulated the collagen III gene. Application of miRNA treatment to modulate TGF-beta expression holds great promise in preventing tendon adhesion formation.

Tendon healing in vivo: gene expression and production of multiple growth factors in early tendon healing period.

PURPOSE: The actions of growth factors during healing of injured flexor tendons are not well characterized, although information pertinent to some individual growth factors is available. We studied gene expression and protein production of a number of growth factors at several time points during the early healing period in a chicken model. METHODS: Seventy-four long toes of 37 white Leghorn chickens were used. The flexor digitorum profundus tendons of 60 toes were surgically repaired after complete transection and were harvested for analysis 3, 5, 7, 9, 14, and 21 days after surgery. The expression of 6 growth factors was studied at 4 time points after surgery with real-time quantitative polymerase chain reactions, and production and distribution of 3 growth factors at all 6 time points were studied by immunohistochemical staining with antibodies. Fourteen tendons that had no surgery served as day 0 controls. Tendon healing status was also assessed histologically. RESULTS: Throughout the early tendon healing period, connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-beta) showed high levels of gene expression. Levels of gene expression of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) were high or moderately high. Expression of the TGF-beta gene was upregulated after injury, whereas the basic fibroblast growth factor (bFGF) gene was downregulated at all postsurgical time points and expressed at the lowest levels among 6 growth factor genes 2 to 3 weeks after surgery. The platelet-derived growth factor B (PDGF-B) gene was also minimally expressed. Findings of immunohistochemistry corresponded to TGF-beta, bFGF, and IGF-1 gene expression. CONCLUSIONS: In this model, up to 3 weeks after surgery, gene expression and production of TGF-beta are high and are upregulated in this healing period. However, expression of the bFGF gene and protein is low and decreases in the healing tendon. The CTGF, VEGF, and IGF-1 genes are expressed at high or moderately high levels, but PDGF-B is minimally expressed.

Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons.

Tendon injury during limb motion is common. Damaged tendons heal poorly and frequently undergo unpredictable ruptures or impaired motion due to insufficient innate healing capacity. By basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) gene therapy via adeno-associated viral type-2 (AAV2) vector to produce supernormal amount of bFGF or VEGF intrinsically in the tendon, we effectively corrected the insufficiency of the tendon healing capacity. This therapeutic approach (1) resulted in substantial amelioration of the low growth factor activity with significant increases in bFGF or VEGF from weeks 4 to 6 in the treated tendons (p < 0.05 or p < 0.01), (2) significantly promoted production of type I collagen and other extracellular molecules (p < 0.01) and accelerated cellular proliferation, and (3) significantly increased tendon strength by 68-91% from week 2 after AAV2-bFGF treatment and by 82-210% from week 3 after AAV2-VEGF compared with that of the controls (p < 0.05 or p < 0.01). Moreover, the transgene expression dissipated after healing was complete. These findings show that the gene transfers provide an optimistic solution to the insufficiencies of the intrinsic healing capacity of the tendon and offers an effective therapeutic possibility for patients with tendon disunion.