External Compression Versus Intravascular Injection: A Mechanistic Animal Model of Filler-Induced Tissue Ischemia.

PURPOSE: Soft tissue ischemia is a devastating and unpredictable complication following dermal filler injection. Multiple mechanisms to explain this complication have been proposed, including vascular compression, vessel damage, and intraarterial filler emboli. To elucidate the mechanism of injury, the authors introduce a mouse model, imaged with optical microangiography and laser speckle contrast imaging technologies, to demonstrate in vivo microvascular response to soft tissue and intravascular filler injection. METHODS: To determine the effect of external vascular compression on distal perfusion, the authors attempted to occlude vessels with subcutaneous hyaluronic acid gel (HAG) bolus injections into the pinna of hairless mice. The authors also performed suture ligation of a major vascular bundle. Following these interventions, laser speckle and optical microangiography were performed serially over 1 week follow up. To determine the effect of intravascular HAG injection, the authors devised and validated a novel method of cannulating the mouse external carotid artery for intraarterial access to the pinna vasculature. Using this model, the authors performed intraarterial HAG injections and completed optical microangiography and laser speckle contrast imaging. RESULTS: Despite large HAG bolus injections directly adjacent to vascular bundles, the authors were unable to induce compressive occlusion of the mouse pinna vessels. Vascular occlusion was successfully performed with suture ligation, but optical microangiography and laser speckle contrast imaging confirmed undisturbed distal capillary bed perfusion. With intravascular HAG injection, large segments of pinna showed distinct perfusion reduction along a vascular distribution when compared with preinjection images, most noticeably at the capillary level. CONCLUSIONS: The novel mouse pinna model combining intravascular access and in vivo microvascular perfusion imaging has furthered the understanding of the mechanism of filler-induced tissue ischemia. Distal capillary perfusion was maintained despite external vascular compression. Intraarterial HAG filler injection, however, resulted in large areas of capillary nonperfusion and is the most likely etiology for filler-induced tissue necrosis that is observed clinically.

Changes in cochlear blood flow in mice due to loud sound exposure measured with Doppler optical microangiography and laser Doppler flowmetry.

In this work we determined the contributions of loud sound exposure (LSE) on cochlear blood flow (CoBF) in an in vivo anesthetized mouse model. A broadband noise system (20 kHz bandwidth) with an intensity of 119 dB SPL, was used for a period of one hour to produce a loud sound stimulus. Two techniques were used to study the changes in blood flow, a Doppler optical microangiography (DOMAG) system; which can measure the blood flow within individual cochlear vessels, and a laser Doppler flowmetry (LDF) system; which averages the blood flow within a volume (a hemisphere of ~1.5 mm radius) of tissue. Both systems determined that the blood flow within the cochlea is reduced due to the LSE stimulation.

Tracking dynamic microvascular changes during healing after complete biopsy punch on the mouse pinna using optical microangiography.

Optical microangiography (OMAG) and Doppler optical microangiography (DOMAG) are two non-invasive techniques capable of determining the tissue microstructural content, microvasculature angiography, and blood flow velocity and direction. These techniques were used to visualize the acute and chronic microvascular and tissue responses upon an injury in vivo. A tissue wound was induced using a 0.5 mm biopsy punch on a mouse pinna. The changes in the microangiography, blood flow velocity and direction were quantified for the acute (<30 min) wound response and the changes in the tissue structure and microangiography were determined for the chronic wound response (30 min-60 days). The initial wound triggered recruitment of peripheral capillaries, as well as redirection of main arterial and venous blood flow within 3 min. The complex vascular networks and new vessel formation were quantified during the chronic response using fractal dimension. The highest rate of wound closure occurred between days 8 and 22. The vessel tortuosity increased during this time suggesting angiogenesis. Taken together, these data signify that OMAG has the capability to track acute and chronic changes in blood flow, microangiography and structure during wound healing. The use of OMAG has great potential to improve our understanding of vascular and tissue responses to injury in order to develop more effective therapeutics.

Quantifying optical microangiography images obtained from a spectral domain optical coherence tomography system.

The blood vessel morphology is known to correlate with several diseases, such as cancer, and is important for describing several tissue physiological processes, like angiogenesis. Therefore, a quantitative method for characterizing the angiography obtained from medical images would have several clinical applications. Optical microangiography (OMAG) is a method for obtaining three-dimensional images of blood vessels within a volume of tissue. In this study we propose to quantify OMAG images obtained with a spectral domain optical coherence tomography system. A technique for determining three measureable parameters (the fractal dimension, the vessel length fraction, and the vessel area density) is proposed and validated. Finally, the repeatability for acquiring OMAG images is determined, and a new method for analyzing small areas from these images is proposed.

Estradiol and G1 reduce infarct size and improve immunosuppression after experimental stroke.

Reduced risk and severity of stroke in adult females is thought to depend on normal endogenous levels of estrogen, a well-known neuroprotectant and immunomodulator. In male mice, experimental stroke induces immunosuppression of the peripheral immune system, characterized by a reduction in spleen size and cell numbers and decreased cytokine and chemokine expression. However, stroke-induced immunosuppression has not been evaluated in female mice. To test the hypothesis that estradiol (E2) deficiency exacerbates immunosuppression after focal stroke in females, we evaluated the effect of middle cerebral artery occlusion on infarct size and peripheral and CNS immune responses in ovariectomized mice with or without sustained, controlled levels of 17-beta-E2 administered by s.c. implant or the putative membrane estrogen receptor agonist, G1. Both E2- and G1-replacement decreased infarct volume and partially restored splenocyte numbers. Moreover, E2-replacement increased splenocyte proliferation in response to stimulation with anti-CD3/CD28 Abs and normalized aberrant mRNA expression for cytokines, chemokines, and chemokine receptors and percentage of CD4(+)CD25(+)FoxP3(+) T regulatory cells observed in E2-deficient animals. These beneficial changes in peripheral immunity after E2 replacement were accompanied by a profound reduction in expression of the chemokine, MIP-2, and a 40-fold increased expression of CCR7 in the lesioned brain hemisphere. These results demonstrate for the first time that E2 replacement in ovariectomized female mice improves stroke-induced peripheral immunosuppression.

Role of signal transducer and activator of transcription-3 in estradiol-mediated neuroprotection.

Estradiol is protective in experimental cerebral ischemia, but the precise mechanisms remain unknown. Signal transducer and activator of transcription-3 (STAT3) is a transcription factor that is activated by estrogen, translocates to the nucleus, and induces the transcription of neuroprotective genes, such as bcl-2. We determined whether estradiol increases STAT3 activation in female rat brain after focal cerebral ischemia and whether STAT3 activation contributes to estradiol-mediated neuroprotection against ischemic brain injury. Ovariectomized (OVX) female rats with and without estradiol replacement were subjected to 2 h of middle cerebral artery occlusion (MCAO), and phosphorylated STAT3 (P-STAT3) and total STAT3 (T-STAT3) were quantified by Western blot analysis at 3 and 22 h of reperfusion. STAT3 activation was colocalized with neuronal and survival markers microtubule-associated protein 2 (MAP2) and Bcl-2 using immunohistochemistry. Infarct size was measured at 22 h after MCAO in estradiol-treated OVX animals in the presence and absence of STAT3 inhibitor cucurbitacin I (JSI-124) using 2,3,5-triphenyltetrazolium chloride staining. Estradiol increased P-STAT3 in the ischemic cortex cytosolic fraction at 3 h after MCAO without affecting T-STAT3. This was associated with increased P-STAT3 in the nuclear fraction, which remained elevated at 22 h after MCAO. The nuclear P-STAT3 colocalized with MAP2 and Bcl-2 within the peri-infarct zone. The P-STAT3 inhibitor JSI-124 abolished the protective effect of estradiol without affecting infarct size in untreated OVX rats. We conclude that estradiol increases STAT3 phosphorylation in neurons after MCAO and that STAT3 activation plays an important role in estradiol-mediated neuroprotection.

ERK1/2 is a negative regulator of homeodomain protein Arix/Phox2a.

The homeodomain protein Arix/Phox2a plays a role in the development and maintenance of the noradrenergic cell type by regulating the transcription of genes involved in the biosynthesis and metabolism of noradrenaline. Previous work has shown that Arix/Phox2a is a phosphoprotein, and the phosphorylated form of Arix/Phox2a exhibits poorer DNA-binding activity than does the dephosphorylated form. Here, we demonstrate that Arix/Phox2a is phosphorylated by extracellular signal-related kinase (ERK)1/2 at two sites within the N-terminal transactivation domain. The phosphorylation level of Arix in cultured SH-SY5Y neuroblastoma cells is reduced when cells are treated with the mitogen activated protein kinase kinase 1 (MEK1) inhibitor UO126. Treatment of sympathetic neurons with the MEK1 inhibitor, PD98059, results in an elevation of mRNAs encoding noradrenergic proteins, dopamine beta-hydroxylase (DBH) and norepinephrine transporter (NET), but not tyrosine hydroyxlase (TH). Treatment of neuroblastoma cultures with PD98059 increases the interaction of Arix with DBH and NET genes, but not the TH gene. Together, these results suggest that phosphorylation of Arix by ERK1/2 inhibits its ability to interact with target genes, and that both specificity of expression and modulation by external stimuli are monitored through the same transcription factor.

Ciliary neurotrophic factor suppresses Phox2a in sympathetic neurons.

The cholinergic differentiation factor ciliary neurotrophic factor (CNTF) suppresses noradrenergic properties while inducing cholinergic and peptidergic properties in sympathetic neurons. In the rat, this includes suppression of the noradrenergic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase. Lower enzyme levels result in part from suppression of gene transcription, but the mechanisms are unknown. We found that ciliary neurotrophic factor decreased the transcriptional activator Phox2a in neuroblastoma cells and cultured sympathetic neurons, suggesting that the loss of Phox2a is part of the mechanism by which CNTF suppresses tyrosine hydroxylase and dopamine beta-hydroxylase. Consistent with this model, Phox2a is suppressed in rat cholinergic sympathetic neurons where noradrenergic enzymes decrease, but is not altered in mouse cholinergic neurons where these enzymes remain high.

Cytokine suppression of dopamine-beta-hydroxylase by extracellular signal-regulated kinase-dependent and -independent pathways.

Cholinergic differentiation factors (CDFs) suppress noradrenergic properties and induce cholinergic properties in sympathetic neurons. The CDFs leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) bind to a LIFR.gp130 receptor complex to activate Jak/signal transducers and activators of transcription and Ras/mitogen-activated protein kinases signaling pathways. Little is known about how these differentiation factors suppress noradrenergic properties. We used sympathetic neurons and SK-N-BE(2)M17 neuroblastoma cells to investigate CDF down-regulation of the norepinephrine synthetic enzyme dopamine-beta-hydroxylase (DBH). LIF and CNTF activated extracellular signal-regulated kinases (ERKs) 1 and 2 but not p38 or Jun N-terminal kinases in both cell types. Preventing ERK activation with PD98059 blocked CNTF suppression of DBH protein in sympathetic neurons but did not prevent the loss of DBH mRNA. CNTF decreased transcription of a DBH promoter-luciferase reporter construct in SK-N-BE(2)M17 cells, and this was also ERK-independent. Cytokine inhibition of DBH promoter activity did not require a silencer element but was prevented by overexpression of the transcriptional activator Phox2a. Inhibiting ERK activation increased basal DBH transcription in SK-N-BE(2)M17 cells, and DBH mRNA in sympathetic neurons. Transfection of Phox2a into PD98059-treated M17 cells resulted in a synergistic increase in DBH promoter activity compared with Phox2a or PD98059 alone. These data suggest that CDFs down-regulate DBH protein via an ERK-dependent pathway but inhibit DBH gene expression through an ERK-independent pathway. They further suggest that ERK activity inhibits basal DBH gene expression.