Electron Microscope Characterization of Platelet Activation State Reveals That Wound Closure and P2Y 12 Receptors Are Major Early Determinants of Thrombus Structure in a Venous Puncture Model

Abstract Vascular damage presents in many forms and varying geometries. Nevertheless, the platelet response to endothelial damage to the blood vessel wall, be it through a prick or a full puncture wound, is thought to be staged by a qualitatively similar temporal variance in signaling agonists. For example, endothelial damage in the microvasculature is thought to be initially dominated by thrombin and later by platelet released ADP and thromboxane. The same temporal sequence in signaling has been proposed to exist in a profusely bleeding puncture wound 1. If so, platelet morphology, a gold standard of platelet activation state, could provide a strong readout of temporally distinct signaling effects. Platelet morphology has long been considered to be a reliable indicator of a strong agonist such as thrombin acting through PAR receptors that produces a rounded, pseudopod extending, degranulated, highly adhesive platelet versus weaker agonists such as ADP or thromboxane acting through P2Y 12 receptors to produce a less adhesive, somewhat rounded platelet. A testable prediction of existing hemostasis models is that temporal staging of signaling leads to temporal differences in platelet morphology within the forming/remodeling thrombus. Such hypothesized temporal differences in signaling are clinically significant as they form the basis for hypothesizing phenotypically distinct outcomes for direct acting anti-coagulants (DOACs) affecting thrombin versus anti-platelet drugs affecting P2Y 12, ADP receptors. Advances in imaging, e.g., wide area transmission electron microscopy (WA-TEM), make possible the local determination of platelet activation state with high precision 2. Taking a mouse jugular vein puncture wound model 1,2, we found that all morphologically recognized platelet activation states were present early, 1 min post puncture, with loosely bound discoid shaped platelets being the most peripherally located. For bleeding, early-stage puncture wound, these loosely adherent, low activation state platelets were located on both intravascular and extravascular thrombus aggregates. Once the puncture wound is closed, loosely adherent platelets were only found on the intravascular surfaces of the thrombus. We propose that this result is most consistent with a platelet conversion model in which new loosely adherent platelets rapidly convert to tightly packed platelets. As the thrombus remodels, 5 and 20 min post-puncture, the thrombus continued to accumulate platelets both intravascularly and extravascularly. Peripheral, discoid shaped platelets provided a source for intravascular thrombus growth. However, any subsequent extravascular thrombus growth must be due to platelet migration. Significantly, we found that cangrelor, a direct acting P2Y 12 inhibitor, stalled thrombus formation/remodeling at an early stage (Figure 1A,C,E see also ref 1,2). By WA-TEM, the accumulation of discoid-shaped, loosely adherent platelets appeared to be enhanced in a cangrelor treated 5 min thrombus (Figure 1E,F). We suggest that P2Y 12 receptors must act early in thrombus formation with the conversion of discoid to more activated platelets being most affected. In contrast, a 5-min post puncture dabigatran (DOAC) treated showed deformed architecture with inhibition of the accumulation of discoid shaped platelets/rounded loosely adherent platelets being most affected (Figure 1D,F, see also ref 2). Accumulation of degranulated platelets appeared to be lessened in both cangrelor and dabigatran treated thrombi. We propose that the simplest explanation of these results is that multiple signaling pathways act in parallel with select activation states being more dependent on one pathway than another. Clinically, our results suggest that P2Y 12 inhibitors can affect thrombus formation at early time points in addition to the late time points projected by current models. 1. Tomaiuolo M., Matzko C.N., Posentud-Fuentes I., Weisel J.W., Brass L.F. & Stalker T.J. Interrelationships between structure and function during the hemostatic response to injury. Proc Natl Acad Sci USA. 116. 2243-2252 (2019). 2. Rhee, Pokrovskaya I.D.,BallK., LingK., VedanapartiY., CohenJ., CruzD., ZhaoO.S., AronovaM.A., ZhangG., Kamykowski J.A., LeapmanR.D., & StorrieB. Venous puncture wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates. Commun. Biol., accepted. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Scanning Electron Microscopy Study of Endothelial Injury and Thrombus Formation in WT and VWF Deficient Mice.

Blood ◽

10.1182/blood.v114.22.3062.3062 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3062-3062

Author(s):

Justin Barr ◽

Jennifer Barr ◽

Marielle Meurice ◽

David Motto

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Thrombus Formation ◽

Endothelial Damage ◽

Wild Type ◽

Subsequent Formation ◽

Time Points ◽

Endothelial Surface ◽

Deficient Mice ◽

Scanning Electron

Abstract Abstract 3062 Poster Board II-1038 VWF is a large plasma glycoprotein required for normal hemostasis, and performs its function through binding to coagulation Factor VIII, and via interactions with both platelet surface glycoproteins and the activated and/or damaged vascular surface. We have developed a scanning electron microscopy (SEM) protocol to visualize endothelial damage and thrombus formation in wild-type and VWF-deficient mice. Thrombus formation is initiated by ferric chloride, and subsequently at defined time points, the circulation is rapidly flushed and aldehyde fixed. The carotid artery is removed, externally fixed, sectioned (both longitudinally and in cross-section), processed for SEM, and visualized. With this protocol we have obtained high-quality images (exceeding 100,000x) of FeCl3-induced endothelial damage and thrombus formation in C57BL/6 and VWF-deficient mice at baseline, and at 30, 60, 90, 120, 240, and 300 seconds post-injury (please access http://sites.google.com/site/mottolab/ to view images). Interestingly, we find that FeCl3 induces little, if any, endothelial denudation and collagen exposure at these time points, with the endothelium clearly appearing changed from baseline, but not damaged. Thus, initial platelet adhesion seems to be occurring in the absence of collagen exposure in this model. In wild-type mice, platelets adhere rapidly to the endothelial surface and assume a cross-linked appearance by 90 seconds, with continual inward growth of the thrombus through the 300 second time point. In VWF-deficient mice, platelets also adhere rapidly to the endothelial surface, but in contrast, remain recognizable longer without assuming a highly-activated phenotype. Compared with wild-type, at all time points examined the VWF-deficient thrombus appears smaller with considerably less cross-linking and platelet activation. Interestingly, during the course of these experiments we also have identified what appears to be red blood cells (RBCs) participating in thrombus formation. Similar to platelets, RBCs interact directly with the endothelial surface, and subsequently become elongated in the direction of blood flow. These elongated RBCs are often observed to cluster and bind platelets, with the subsequent formation of large platelet-erythrocyte complexes. Further characterization of these complexes and the role they may play in thrombus formation is currently in progress. Additionally, similar SEM studies are underway with both ADAMTS13-deficient and GPIb alpha-deficient mice, and with mice transiently expressing in vivo biotinylated VWF for visualization of this molecule at high magnification and resolution. These studies should help better define the mechanisms of endothelial activation and thrombus formation as they occur in situ. Disclosures No relevant conflicts of interest to declare.

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GRK6 Regulates the Hemostatic Response to Injury through Its Rate-Limiting Effects on GPCR Signaling in Platelets

Blood ◽

10.1182/blood-2019-129175 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3611-3611

Author(s):

Xi Chen ◽

Shuchi Gupta ◽

Matthew Cooper ◽

Daniel Dehelian ◽

Xuefei Zhao ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Early Stage ◽

Thrombus Formation ◽

Surface Expression ◽

Cerebrovascular Diseases ◽

Human Platelets ◽

Akt Activation ◽

Granule Secretion ◽

First Time

Inappropriate platelet activation remains a major cause of cardiovascular and cerebrovascular diseases. Most agonists activate platelets through G protein-coupled receptors (GPCRs). However, questions remain about mechanisms that provide negative feedback towards activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in WT controls during the early stages of thrombus formation, with a rapid increase of platelet accumulation at site of injury. Platelet activation in the absence of GRK6 is enhanced, but in an agonist-selective manner. Responses to PAR4 agonist peptide or ADP stimulation in GRK6-/- platelets are increased compared to WT control littermates, while the response to TxA2 is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that in human platelets, platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase of the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Collectively, these observations, for the first time, show that 1) GRK6 regulates the hemostatic response to injury by thrombin and ADP, 2) it mediates platelet activation by reducing PAR1/4- and P2Y12-dependent signaling, and 3) GRK6 limits the rate of platelet activation during early stage of thrombus growth and helps prevent inappropriate platelet activation. Disclosures No relevant conflicts of interest to declare.

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Role of the Platelets and Nitric Oxide Biotransformation in Ischemic Stroke: A Translative Review from Bench to Bedside

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/2979260 ◽

2020 ◽

Vol 2020 ◽

pp. 1-18

Author(s):

Maciej Bladowski ◽

Jakub Gawrys ◽

Damian Gajecki ◽

Ewa Szahidewicz-Krupska ◽

Anna Sawicz-Bladowska ◽

...

Keyword(s):

Nitric Oxide ◽

Ischemic Stroke ◽

Platelet Activation ◽

Thrombus Formation ◽

Endothelial Damage ◽

Guanosine Monophosphate ◽

No Production ◽

Nos Inhibitors ◽

No Bioavailability

Ischemic stroke remains the fifth cause of death, as reported worldwide annually. Endothelial dysfunction (ED) manifesting with lower nitric oxide (NO) bioavailability leads to increased vascular tone, inflammation, and platelet activation and remains among the major contributors to cardiovascular diseases (CVD). Moreover, temporal fluctuations in the NO bioavailability during ischemic stroke point to its key role in the cerebral blood flow (CBF) regulation, and some data suggest that they may be responsible for the maintenance of CBF within the ischemic penumbra in order to reduce infarct size. Several years ago, the inhibitory role of the platelet NO production on a thrombus formation has been discovered, which initiated the era of extensive studies on the platelet-derived nitric oxide (PDNO) as a platelet negative feedback regulator. Very recently, Radziwon-Balicka et al. discovered two subpopulations of human platelets, based on the expression of the endothelial nitric oxide synthase (eNOS-positive or eNOS-negative platelets, respectively). The e-NOS-negative ones fail to produce NO, which attenuates their cyclic guanosine monophosphate (cGMP) signaling pathway and—as result—promotes adhesion and aggregation while the e-NOS-positive ones limit thrombus formation. Asymmetric dimethylarginine (ADMA), a competitive NOS inhibitor, is an independent cardiovascular risk factor, and its expression alongside with the enzymes responsible for its synthesis and degradation was recently shown also in platelets. Overproduction of ADMA in this compartment may increase platelet activation and cause endothelial damage, additionally to that induced by its plasma pool. All the recent discoveries of diverse eNOS expression in platelets and its role in regulation of thrombus formation together with studies on the NOS inhibitors have opened a new chapter in translational medicine investigating the onset of acute cardiovascular events of ischemic origin. This translative review briefly summarizes the role of platelets and NO biotransformation in the pathogenesis and clinical course of ischemic stroke.

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Endothelial Injury and Thrombus Formation In the Ferric Chloride Murine Model of Hemostasis

Blood ◽

10.1182/blood.v116.21.650.650 ◽

2010 ◽

Vol 116 (21) ◽

pp. 650-650 ◽

Cited By ~ 1

Author(s):

Justin Barr ◽

Anil Chauhan ◽

David G Motto

Keyword(s):

Carotid Artery ◽

Ferric Chloride ◽

Murine Model ◽

Thrombus Formation ◽

Endothelial Damage ◽

Endothelial Injury ◽

Time Points ◽

Size And Shape ◽

Endothelial Denudation

Abstract Abstract 650 Laboratory mice are used in hemostasis and thrombosis research in experiments ranging from the investigation of basic mechanisms of thrombus formation to the generation of preclinical data regarding potential therapeutic anti-thrombotic agents. With the common ferric chloride (FeCl3) model of vascular thrombosis, FeCl3 is applied directly to outside of the carotid artery or mesenteric vessels to induce thrombus formation. However, despite its widespread use, surprisingly little is understood regarding the mechanisms by which FeCl3 induces endothelial injury and subsequent thrombus formation, although it is believed that endothelial denudation and collagen exposure occur, and trigger the onset of thrombosis. Similarly, very little is known regarding the nature of the thrombi that are generated by this method, and importantly, whether these experimental thrombi resemble bona-fide thrombi that occur as a result of “true” vascular injury. To address these issues, we developed a scanning electron microscopy (SEM) protocol to visualize endothelial damage and thrombus formation that occur in situ. Briefly, thrombus formation is initiated by direct application of FeCl3 (or vehicle control) to the carotid artery, and subsequently at defined time points, the circulation is flushed, and then internally aldehyde-fixed. The affected section of carotid artery is removed, externally fixed, sectioned, processed for SEM, and visualized. With this procedure, we have obtained high-quality and high-magnification images of FeCl3-induced endothelial damage and thrombus formation through approximately 5 minutes of injury, in multiple samples and uninjured controls (N>100). Interestingly, we found that through these time points, FeCl3 induces little, if any, endothelial damage or subendothelial exposure. Rather, the endothelium rapidly assumes an “activated” phenotype, clearly changed from baseline, but non-denuded and intact. For verification, we found that mechanical injury of the murine carotid results in endothelial denudation which is easily identified by SEM. Perhaps more surprisingly, we also found that the first cells to adhere to the endothelium following FeCl3 application actually are red blood cells (RBCs), and not platelets. Following binding to the vascular surface, RBCs become misshapen and elongated in the direction of blood flow, or break off, leaving fragments associated with the endothelial surface. Subsequently, surface-bound RBCs and fragments bind additional RBCs and platelets from the circulation, forming large and characteristic-appearing complexes. From this point forward, large numbers of additional platelets accumulate on the RBC and platelet/RBC complexes, and the thrombus grows inward rapidly until the vessel becomes occluded. Next, to investigate formation of these potential RBC and platelet/RBC complexes with an independent system, we turned to intravital fluorescent microscopy in mesenteric venules. We found that FeCl3 rapidly induces structures consistent in size and shape with the “early” platelet/RBC complexes observed by SEM. With further time points, these small/early lesions subsequently bound more labeled platelets, and increased rapidly in size until occlusion of the vessel. As these platelet lesions grew, they also resembled in size and shape the “later” platelet/RBC complexes observed by SEM, including possession of a characteristic trailing “tail” of platelets. It is interesting that similar structures appeared to form in both the carotid artery and mesenteric venules, given the marked difference in shear stress between the two (1000-1500 s-1 vs. 100–200 s-1, respectively). To our knowledge, the existence of such platelet/RBC complexes has not been documented in any system. In summary, we have developed a technique to investigate in situ endothelial damage and thrombus formation in mice by SEM, and demonstrated that thrombus formation in the commonly-used FeCl3 murine model occurs in the absence of endothelial denudation, and appears to involve the active participation of RBCs. Further work will be necessary to confirm whether or not RBCs are necessary for thrombus formation in this model, and whether RBCs participate in any type of naturally-occurring hemostasis or thrombosis. Additionally, these results will likely have strong impact on future interpretation of experiments resulting from the use of FeCl3 in mice. Disclosures: No relevant conflicts of interest to declare.

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Platelet Activation State Intermixing in a Venous Puncture Model Indicates Novel Patterns of Thrombus Formation

Blood ◽

10.1182/blood-2019-130805 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 9-9

Author(s):

Brian Storrie ◽

Sung W. Rhee ◽

Irina D Pokrovskaya ◽

Kenny Ling ◽

Yajnesh Vedanaparti ◽

...

Keyword(s):

Electron Microscopy ◽

Platelet Activation ◽

Vessel Wall ◽

Jugular Vein ◽

Thrombus Formation ◽

Sem Images ◽

Puncture Wound ◽

Activated Platelets ◽

Puncture Model ◽

Scanning Electron

Introduction: Platelet recruitment to generate a thrombus is key to bleeding cessation. That recruitment is dependent on a series of platelet activation processes that include adhesion to the exposed vessel matrix, platelet-platelet adhesion and platelet granule release. How platelet activation is patterned to generate a thrombus has previously been studied by intravital light microscopy, two-photon microscopy and scanning electron microscopy at resolutions insufficient to infer platelet activation at the level of the individual platelet. Here, we present a collaborative effort to stratify spatially the extent of platelet activation at the cellular level in a mouse jugular vein puncture model. We used wide-area transmission electron microscopy (WA-TEM) and serial block face scanning electron microscopy (SBF-SEM) at a resolution of 3 to 100 nm across whole thrombi to determine activation state of individual platelets. Our results, indicate a pattern of platelet stratification within the puncture wound that varies in a time-dependent manner with distinct structural stages in the formation of the thrombus. Methods: Jugular vein thrombi from C57BL/6 mice were collected 1, 5, or 20 min after a 300 µm needle puncture and prepared for EM imaging. For WA-TEM, hundreds of overlapping 3 nm resolution images were acquired using a gondimeter stage. The images sets were aligned using NIH Fiji software to create a single high-res, thrombus-wide image. Individual platelets were stratified into morphologically defined activation states (1: no activation, 2: decreased granule number, 3: no visible granules left, 4: hollow inside). The spatial distribution of platelet stratification was analyzed using iVision software. For SBF-SEM, 100-nm XY-resolution SEM images were collected every 200 nm and 20 nm XY-resolution images every 20 µm. Semi-automated stratification of platelet activation state in individual slices of the thrombus were combined into a 3-D representation using Amira software. Volumetric distributions of platelets with respect to the puncture hole and the vascular wall were quantified. Results: Thrombus Formation Stage 1 (anchor and extend) -- One min post puncture, platelets were anchored in clumps along the exposed vessel wall. Near the damaged vessel wall was a peripheral layer of activated or degranulated platelets (states 3 and 4) covered by additional layers of less-activated platelets (state 1 and 2). Short cylindrical ingrowths extended into the 300 µm hole. Unexpectedly, large aggregates of platelets with a mixture of activation states (states 1 - 4) were found extending from these anchor points into the hole and vertically into the intravascular space. Aggregate surface layers were composed mostly of degranulated platelets (states 3 and 4). Less than 40% of neighboring platelets were of the same activation state as their neighbor. Surprisingly, <2% of platelet-occupied volume within the puncture hole contained largely degranulated platelets (aggregates of only states 3 and 4). Stage 2 (cap and erect) -- At 5 min after injury, the puncture hole was capped. ~70% of platelets neighboring degranulated platelets (3 and 4) formed visible aggregates within the thrombus. These aggregates were found along the exposed vessel wall and encasing vertical platelet aggregate towers containing a mixture of platelets in different states (1 - 4). Towers were typically separated by large cavities. SBF-SEM images, a machine-based, unbiased sampling of the underlying platelet distribution, revealed that ~10% of the platelet volume in the puncture hole of the thrombus and the intravascular towers contained largely degranulated platelets, similar to the data from WA-TEM. Stage 3 (infill and remodeling) - At 20 min post-puncture, the thrombus was filled with a mixture of platelets of varying activation states, which surrounded central, vertical aggregates (towers) of degranulated platelets seen at 5 min. Only minor cavity space was apparent. The intravascular surface of the thrombus was covered with an ~10 platelet-thick layer of loosely packed, variably activated platelets (states 1 - 4). Conclusions: Our results demonstrate dynamic spatial patterns of platelet activation within a forming puncture-wound thrombus. Such patterns yield insights into thrombus formation and suggest the need to reference platelets defects and anti-thrombotics drugs against new models. Figure Disclosures No relevant conflicts of interest to declare.

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Duffy antigen / receptor for chemokines correlates with inflammatory reaction in rats with venous hypertension: implication for the pathogenesis of primary chronic venous disease

VASA ◽

10.1024/0301-1526/a000327 ◽

2014 ◽

Vol 43 (1) ◽

pp. 47-54 ◽

Cited By ~ 1

Author(s):

Weibin Huang ◽

Weiwei Qin ◽

Lei Lv ◽

Haoyv Deng ◽

Hao Zhang ◽

...

Keyword(s):

Mononuclear Cells ◽

Early Stage ◽

Antigen Receptor ◽

Venous Hypertension ◽

Skin Tissue ◽

Chronic Venous Disease ◽

Venous Disease ◽

Dependent Manner ◽

Time Points ◽

Duffy Antigen

Background: Duffy antigen / receptor for chemokines (DARC) possesses high affinity for several chemokine subgroups of CC and CXC. Although DARC has been shown to play a role in many inflammatory diseases, its effect on chronic venous disease (CVD) remains unidentified. We explored whether the expression of DARC in skin tissue was activated under venous hypertension as well as the relationships between DARC and inflammation. Materials and methods: The inflammation in a rat model of venous hypertension caused by a femoral arterial-venous fistula (AVF) was studied. At specified intervals the pressure in the femoral veins was recorded within 42 days. Hindlimb skin specimens were harvested at different time points. The expressions of DARC, interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) in skin tissue were examined. Mononuclear cells infiltrated in skin tissue were detected. Results: Femoral venous pressures in AVF groups increased significantly at different time points (P < 0.01). DARC was expressed in skin tissue and its expression level increased significantly in AVF groups from the 7nd day on and was enhanced in a time-dependent manner within 42 days (P < 0.05). Meanwhile, both MCP-1 and IL-8 had higher levels, accompanied by increased mononuclear cells infiltrating into skin tissue (P < 0.05). Conclusions: A rat AVF model which can maintain venous hypertension for at least 42 days is competent for researching the pathogenesis of CVD. DARC, which plays a role in the inflammation of skin tissue under venous hypertension, may become a new molecular target for diagnosis and treatment of CVD at a very early stage.

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An Electron Microscopic Study of Platelet Thrombus Formation in the Rabbit with Particular Regard to 5-Hydroxytryptamine Release

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655068 ◽

1967 ◽

Vol 18 (03/04) ◽

pp. 592-604 ◽

Cited By ~ 15

Author(s):

H. R Baumgartner ◽

J. P Tranzer ◽

A Studer

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Vessel Wall ◽

Thrombus Formation ◽

Endothelial Damage ◽

Electron Microscopic ◽

Rabbit Ear ◽

Basement Lamina ◽

Platelet Thrombus

SummaryElectron microscopic and histologic examination of rabbit ear vein segments 4 and 30 min after slight endothelial damage have yielded the following findings :1. Platelets do not adhere to damaged endothelial cells.2. If the vessel wall is denuded of the whole endothelial cell, platelets adhere to the intimai basement lamina as do endothelial cells.3. The distance between adherent platelets as well as endothelial cells and intimai basement lamina measures 10 to 20 mµ, whereas the distance between aggregated platelets is 30 to 60 mµ.4. 5-hydroxytryptamine (5-HT) is released from platelets during viscous metamorphosis at least in part as 5-HT organelles.It should be noted that the presence of collagen fibers is not necessary for platelet thrombus formation in vivo.

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N-Acetylcysteine Added to Local Anesthesia Reduces Scar Area and Width in Early Wound Healing—An Animal Model Study

International Journal of Molecular Sciences ◽

10.3390/ijms22147549 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7549

Author(s):

Wiktor Paskal ◽

Adriana M. Paskal ◽

Piotr Pietruski ◽

Albert Stachura ◽

Kacper Pełka ◽

...

Keyword(s):

Wound Healing ◽

Early Stage ◽

Healing Process ◽

Wound Healing Process ◽

Sprague Dawley ◽

Time Points ◽

Model Study ◽

Sprague Dawley Rats ◽

Animal Model Study ◽

Anesthetic Solution

The aim of the study was to evaluate if a pre-incisional N-acetylcysteine (NAC) treatment altered the process of wound healing in a rat model. The dorsal skin of 24 Sprague-Dawley rats was incised in six locations. Before the incisions were made, skin was injected either with lidocaine and epinephrine (one side) or with these agents supplemented with 0.015%, 0.03%, or 0.045% NAC (contralaterally). Photographic documentation of the wound healing process was made at 11 time points. Rats were sacrificed 3, 7, 14, or 60 days after incision to excise scars for histological analysis. They included: Abramov scale scoring, histomorphometry analysis, and collagen fiber arrangement assessment. Skin pretreated with 0.03% NAC produced the shortest scars at all analyzed time points, though this result was statistically insignificant. At this NAC concentration the scars had smaller areas on the third day and were narrower on the day 4 compared with all the other groups (p < 0.05). On day 7, at the same concentration of NAC, the scars had a higher superficial concentration index (p = 0.03) and larger dermal proliferation area (p = 0.04). NAC addition to pre-incisional anesthetic solution decreased wound size and width at an early stage of scar formation at all concentrations; however, with optimal results at 0.03% concentration.

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