Pathological and Immunohistochemical Assessment of Aging of the Abdominal Skin Treated with Carboxytherapy: A Randomized, Split-body Trial

J Clin Aesthet Dermatol. 2024;17(8):62–69.

by Nooshin Bagherani MD, MA, PhD Candidate; Alireza Ghanadan, MD; Golshan Mirmomeni, MA; Alireza Firooz, MD; Bruce R. Smoller, MD; Reza Shojaei, MD; Haniyeh Rafipour, MD; Negin Bagherani, bS; Mansoreh Abdolhosseini, PhD Candidate; and Gholamreza Tavoosidana, PhD

Dr. Bagherani is with the Department of Molecular Medicine, School of Advanced Technologies in Medicine at the Tehran University of Medical Sciences in Tehran, Iran. Dr. Ghanadan is with the Department of Dermatopathology, Razi Hospital and Department of Pathology, Cancer Institute at the Imam Khoemini Hospital Complex in Tehran, Iran. Mrs. Mirmomeni is with the Hearing Research Center at the Ahvaz Jundishapur University of Medical Sciences in Ahvaz, Iran. Dr. Firooz is with the Center for Research & Training in Skin Diseases & Leprosy, Clinical Trial Center at the Tehran University of Medical Sciences in Tehran, Iran. Dr. Smoller is with the Department of Pathology at the University of Rochester School of Medicine and Dentistry in Rochester, NY. Dr. Shojaei is with the Department of Surgery at the Arak University of Medical sciences in Arak, Markazi Province, Iran. Dr. Rafipour is with the Medical School at the Tehran University of Medical Sciences in Tehran, Iran. Dr. Bagherani is with the Department of Cellular Biology, Payam-e-Noor University of Arak in Arak, Iran. Drs Abdolhosseini and Tavoosidana are with the Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences in Tehran, Iran.

FUNDING: This article was funded by the Tehran University of Medical Sciences and Health Services.

DISCLOSURES: This study was supported by Tehran University of Medical Sciences in partial fulfillment of the requirement of PhD of molecular medicine. Nik Fannavaran Plasma Co., supported through renting the carboxytherapy machine and accepting machine service-related fees.

ABSTRACT: Background. Skin aging as a continuous and irreversible process is mainly the result of alterations of function and structure of the dermis. Among the modalities used for treating skin aging, carboxytherapy has been introduced as a safe minimally-invasive method for rejuvenation, reparation, and reconditioning of the skin.

Objective. We assessed the efficacy of carboxytherapy for the treatment of intrinsic skin aging through pathological and immunohistochemical (IHC) investigations.

Methods. Our study was a split-body, randomized clinical trial on 15 female patients with intrinsic skin aging of the abdomen. Carboxytherapy was performed on one side of the abdomen, weekly for 10 sessions, while the other side was left untreated. Two weeks after the last session, skin biopsies were taken from both sides of the abdomen. Staining with hematoxylin-eosin, Masson-trichrome, and Orcein Giemsa was performed for the assessment of epidermal and dermal thickness, collagen, and elastin organization, respectively. IHC examination was performed for investigation of TGF-β1 and VEGF.

Results. Pathological examination showed a significant increase in epidermal and dermal thickness and re-organization of collagens and elastic fibers with carboxytherapy. IHC examinations revealed a significantly increased expression of TGF-β1 and VEGF with carboxytherapy.

Conclusion. Our study demonstrated the effectiveness of carboxytherapy in treating and reversing intrinsic aging skin through pathological and IHC studies.

Keywords. carboxytherapy, skin aging, pathology, immunohistochemistry

Introduction

Aging as a continuous and irreversible process affecting the skin is clinically characterized by skin laxity, wrinkling, fragility, xerosis, and pruritus, increased sensitivity to temperature, impaired wound healing, and increased risk of malignancies.³ This process, which mainly is the result of alterations of function and structure of the dermis, is characterized by thinning, decreased hydration, and loss of elasticity.⁴ On pathological examination, the epidermis progressively becomes thinner and rete ridges become less pronounced with aging. Aging-related uneven distribution of the pigment is due to disruption of the effective transfer of melanin pigment from the melanocytes to the keratinocytes with prolonged ultraviolet (UV) exposure.⁵ The normal dermis, which is mainly comprised of the type I collagen in the reticular dermis and the type III collagen in the papillary dermis, is influenced by aging largely center around UV-induced damage. The type VII collagen is also reduced with aging leading to the clinical appearance of wrinkling.⁶ The elastic tissue fibers, as another component of the dermal connective tissue, are composed of two components including oxytalin and elaunin. In the intrinsic skin aging process, a progressive loss of these fibers is seen ultimately leading to their near absence in the skin of the elderly.⁷ With aging, due to diminished extravascular support and long-standing intraluminal pressure, progressive vascular ectasia, thinning and fragility of the vessel walls and hemorrhage are observed.⁸

The skin aging as a complex process results from intrinsic factors with genetic basis and extrinsic factors due to detrimental environmental factors, mainly UV radiation.⁴ The intrinsic skin aging is an inevitable physiological process which results in fine wrinkles, xerosis, and dermal atrophy, whereas the extrinsic one leads to coarse wrinkles, rough appearance, laxity, and impaired skin elasticity.⁹ 

Compared to young skin, skin of the elderly is more prone to a variety of dermatoses; 13.3 percent of skin conditions have been reported in persons over 60 years of age. Additionally, noticeable association between systemic disorders and skin problems in general population necessitates special notice to skin aging.³ 

The main aim of the management of aging is improvement of the life quality and prevention of age-related conditions in the elderly. Regarding skin aging, its management is particularly important because the skin shows the most clinically noticeable features of the aging mechanism and the status of one’s health.¹⁰

Many different medical and procedural approaches have been introduced for the management of skin aging. Among these modalities, carboxytherapy has been introduced as a safe minimally invasive method for rejuvenation, reparation, and reconditioning of the skin.¹¹ It acts through vasodilatation^11–14, increased permeability of the capillary walls¹³, stimulation of dermal fibroblasts leading to production of higher quantities and higher quality collagen and elastin¹⁵, increased collagen remodeling¹⁴, increased flexibility and decreased firmness of collagen fibers¹³, improved tissue drainage¹², release of  local growth factors in relation to angiogenesis^12,15, lipolysis, and skin regeneration¹⁵, improved trophicity of the treated body site, and  reduction of adipose tissue.¹²

Regarding efficacy of carboxytherapy in the treatment of skin aging, the studies are restricted to a few studies with only a limited number of cases. In the current studies, we assessed the efficacy of carboxytherapy in management of intrinsic skin aging through pathological and immunohistochemical (IHC) studies. 

Methods

We conducted a prospective right-left sided randomized clinical trial and as a portion of a larger project with the ethical code: IR.TUMS.MEDICINE.REC.1400.1077, and clinical registry code: ChiCTR2200055185. The study was completed in the Department of Dermatology at Arak University of Medical Sciences. 

Participants. Exclusion and inclusion criteria. Male and female individuals older than 40 years with visible aging of the abdominal skin, including wrinkles, laxity, pigmentary alteration, and striae distensae, open to inclusion in the study. Participants were excluded if they had active skin infections at the site of biopsy, connective tissue diseases or genodermatoses, keloid tendency, or any systemic disorder, including diabetes mellitus, kidney diseases, cardiac diseases, respiratory diseases, liver diseases, and severe anemia. Smokers and pregnant and/or lactacting women were also excluded, in addition to those taking any medications or supplements within six weeks of the study initiation or those who had undergone any anti-aging treatment or procedure at the site of study within one year prior to study initiation.

Sample size. Sample size was calculated by G*Power with considering pathological variables as qualitative variables and reference to a study by Oliveira et al.¹⁶ (Table 1). 

Study procedure. After getting written informed consent from all participants, carboxytherapy was done on one side (case group), and the other side was left untreated (control group). In each subject, the case and control groups were selected randomly through a coin flip (Figure 1). Randomization was done by the department secretary, who was blinded to the process of the study.

In our study, a digitally controlled carboxytherapy device (“MEDAION”, Nik Fannavaran Plasma Co., Tehran, Iran) was applied. CO₂ was injected intradermally on the treated side in the volume of 150 cc with following parameters: input pressure of 238 kPa, output pressure of 50 kPa, volume per shot of 2.0 cc, and gas temperature of 40°C. The treatment was performed weekly for 10 sessions for constant treatment and control sides. 

Two weeks after the last session of carboxytherapy, skin specimens were taken by 5mm sterile and disposable punches from the right and left side of the abdomen for the pathological and IHC investigations. The samples were fixed in 10% formalin, embedded in paraffin, and sectioned with microtome at 4 to 5 microns. 

For pathological examination, the specimens were stained with hematoxylin-eosin, Masson-trichrome, and Orcein Giemsa. Epidermal and dermal thickness of the specimens was assessed by two dermatopathologists blinded to the process of study based upon observing the hematoxylin-eosin-stained sections. The distance between the top of the granular cell layer and the dermo-epidermal junction at the deepest part was considered as the epidermal thickness.¹⁷ The dermal thickness was defined as the distance between the dermo-epidermal junction at the highest part and the dermo-subcutis junction.  

Masson-trichrome and Orcein Giemsa were used for assessment of the collagen and elastin status, respectively.¹⁶ The organization of these 

fibers was evaluated based using an objective scoring system (1= Very poorly organized; 2=Poorly organized; 3=Monderately organized; 4=Well organized, 5=Very well organized). This assessment was performed by the same dermopathologists blinded to the project.

For the IHC study, we assessed TGF-β1 and VEGF. Primary antibodies used in our study include: 1-TGF-β1 (3C11) (sc-130348; SANTA CRUZ BIOTECHNOLOGY, INC.): A murine monoclonal antibody raised against the recombinant TGF-β1 of human origin and 2-VEGF (C-1) (sc-7269; SANTA CRUZ BIOTECHNOLOGY, INC.): A murine monoclonal antibody raised against the amino acids 1–140 of VEGF of human origin. 

In our study the secondary antibody was from Master Diagnostica (Granada, Spain). The IHC assessment was performed by the same blinded dermatopathologists based upon a scoring system (0=0% staining intensity; 1=<10%; 2=10%–29%; 3=30%–59%; 4=60%–100%).¹⁸

Results

Thirty samples taken from 15 subjects were studied through pathological and IHC assessments. All 15 participants were female. Average age of the participants was 44.43 (±4.29) years. The minimum and maximum ages were 40 and 56.5 years, respectively. Carboxytherapy was performed on the right side in 8 (53.3%) subjects and on the left side in 7 (46.7%) patients. 

Based upon examination of hematoxylin-eosin stained-slides (Figure 2), the mean epidermal thickness was 0.25mm (±0.06) and 0.14mm (±0.02) in the interventional and control groups, respectively.  The mean dermal thickness was 3.2mm (± 1.02) and 1.5mm (± 0.8) in the interventional and control groups respectively. Based upon the paired t-test, comparison of the mean thickness of epidermis and dermis between the studied groups showed the increase in the epidermal and dermal thickness was statistically significant in the interventional group (P<0.001). Figures 3 and 4 show the epidermal and dermal thickness of the interventional and control groups for comparison.

Assessment of the collagen’s status via examination of Masson-trichrome-stained slides (Figure 5) showed that in the interventional group, 60 percent of the subjects had a score of 4, and 40 percent of the cases had a score of 5; whereas, in the control group, 60 percent of cases scored 3, and 40 percent of cases scored 4 (Figure 6). Comparison of collagen scores between the study groups based on NPar Wilcoxon Signed Ranks Test revealed that this score was significantly higher in the interventional group.

Evaluation of the elastin status with Orcein Giemsa (Figure 7) staining demonstrated that in the interventional group, the highest frequency (66.7%) belonged to score 4; whereas, in the control group, score 3 showed the highest frequency (73.3%) (Figure 8). Comparison of the elastin scores between the two studied groups based on NPar Wilcoxon Signed Ranks Test revealed that this score was significantly higher in the interventional group.

Assessment of the epidermal TGF-β1 in the interventional group showed the highest frequency (86.7%) for score 3; whereas, in the control group, the highest frequency (93.3%) was observed for score 2 (Figure 9). Comparing the scores of this growth factor between the studied groups based on the NPar Wilcoxon Signed Ranks Test revealed that the acquired score was significantly higher in the interventional group (P<0.001).

Evaluation of the lymphocytic TGF-β1 (Figure 10) in the interventional group showed the highest frequency (93.3%) for score 3; whereas, in the control group the highest frequency (66.7%) belonged to score 2 (Figure 11). Comparing the scores of this marker between the 2 studied groups based upon NPar Wilcoxon Signed Ranks Test showed that the score was significantly higher in the interventional group in comparison to the control group (P=0.001).

Assessment of VEGF (Figure 12) in the interventional group showed that all cases scored 1; whereas, its evaluation in the control group showed that 80 percent of cases had a score of zero (Figure 13). Comparison of the scores acquired from the assessment of this angiogenic growth factor between the studied groups based on NPar Wilcoxon Signed Ranks Test revealed that it was significantly higher in the interventional group than the control group (P=0.001).

Discussion

Carboxytherapy is a modality for intradermal and/or subcutaneous microinjections of sterile purified CO₂ into different parts of the body to target therapeutic objectives.^11,12 Its effectiveness has been revealed in a variety of dermatologic and cosmetic conditions including skin aging, androgenetic alopecia, localized fat, cellulite, striae distensae, infraorbital hyperpigmentation, and some autoimmune disorders including vitiligo, psoriasis, alopecia areata, and morphea. In the current study, we evaluated the efficacy of carboxytherapy in management of skin aging using pathological and immunohistochemical studies. Staining with hematoxylin-eosin, Masson-Trichrome, and Orcein Giemsa was used for assessing the histologic parameters of the epidermal and dermal thickness, collagen organization and elastin fibers’ status, respectively. We applied IHC for investigating expression of TGF-β1 and VEGF.

The collagens which are the most abundant proteins in the extracellular matrix (ECM) play a key role in the tensile skin strength of the skin and shape of tissues.¹⁹ Type I collagen, the major component of the ECM, is destroyed by UVB.²⁰ In intrinsically aged skin, its production decreases due to downregulation of the TGF-β/Smad signaling and its downstream connective tissue growth factor.⁹ The elastic fibers, as the other vital component of the ECM with elasticity and resilience properties, are observed in several organs such as the skin, lungs, and blood vessels. With aging, these fibers are altered by a variety of enzymatic, chemical and biophysical factors that lead to the accumulation of formation of damaged fibers.²¹

The TGF-β, as one of the most important regulators of the ECM biosynthesis, controls the homeostasis of the collagens in the fibroblasts via regulation of the collagen production and degradation through the Smad pathway. The genes of collagens, fibronectin, decorin, and versican are directly upregulated by the TGF-β/Smad signaling pathway. Therefore, alteration of this pathway leads to decrease of neocollagenesis in the dermis, and subsequently, reduction of the net collagen content. Production of increased amounts of reactive oxygen species (ROS) in aged skin induces the expression of the matrix metalloproteinases (MMPs) and inhibits the TGF-β signaling, which consequently results in the increased degradation and decreased production of the collagens.²²

The epidermis-derived VEGF as a homodimeric, heparin-binding glycoprotein plays an important role as a potent angiogenic factor.²³ The necessity of this factor has been demonstrated in skin rejuvenation, which has confirmed the “angiogenesis hypothesis of aging”. The cutaneous vascular networks undergo considerable alteration with aging process resulting in a reduced microvasculature.²⁴ These facts show the importance of the VEGF in reverting the skin aging manifestations.   

Regarding the efficacy of carboxytherapy in management and treatment of skin aging, the studies are few. In a study on 60 subjects with facial wrinkles, Nassar  et al.assessed and compared the efficacy of radiofrequency (RF), intense pulsed light (IPL) and carboxytherapy in skin rejuvenation through clinical, pathological and HIC examination. In this clinical trial, 4 sessions of each of these treatments were done within 3- to 4-week intervals. The results were assessed three months after the last session. The authors showed the efficacy of IPL in facial rejuvenation, followed by carboxytherapy and RF, respectively. Upon pathological examination of hematoxylin-eosin-stained skin specimens, a moderate increase in the density and number of the collagen fibers, and moderate increase in the thickness of the dermis and dermoepidermal junction were seen in the IPL and carboxytherapy groups; whereas, in the RF group sections, only mild changes of these variables were observed. The results were confirmed by a larger increase of MMP-1 expression in the group of IPL and carboxytherapy in comparison with RF through IHC examination.²⁵ MMP-1 as a collagenase 1 acts through activating catalyzation of the types I, II, and III collagens leading to destruction of damaged collagens and replacement of the matrix. Hence, it is important in wound healing, skin response to UV radiation, and prevention of aging.²⁶ 

In a quasi-experimental case series on nine women candidates for abdominoplasty, Oliveira et al¹⁶ studied the efficacy of carboxytherapy in management of skin laxity through histological examination of collagen and elastin fibers’ status by staining with Picrosirius red and Verhoeff, respectively. The subjects received one session of treatment in the left side of the infra-umbilical region and the skin specimens were gathered 60 days after the treatment.  Histological evaluation demonstrated an increase in the synthesis of collagen and elastic fibers in the treated site in comparison with the control site. In this study, morphometry revealed a significant increase in the collagen percentage in the interventional group compared to the control site; whereas, the morphometric analysis of the elastic fibers’ percentage showed no significant difference between the two studied groups. The authors recommended carboxytherapy as an effective therapeutic approach for skin laxity.

Pinheiro et al²⁷ compared the effectiveness of carboxytherapy and RF on skin aging in eight subjects after abdominoplasty. In this split-body trial, a single session of carboxytherapy and RF were performed on the right and left infra-umbilical regions, respectively.  The skin samples from each site of the treatment were taken 120 days after treatment, and a skin specimen from the untreated site was considered as a control group. Picrosirus and Weigert staining were used for evaluation of collagen and elastin fibers’ status, respectively. Pathological assessment revealed collagen remodeling in both groups in comparison with the control group; however, this remodeling was more prominent and lasted longer in the RF group and these changes were more subtle in the carboxytherapy group. An increase in elastic fibers was seen with carboxytherapy, whereas no change of elastin fibers was seen with RF.²⁷

In a pilot, prospective, randomized, single-blind clinical trial, Medrano et al²⁸ assessed the objective effectiveness and subjective satisfaction of carboxytherapy gel mask in acceleration of wound healing in nine subjects with facial photoaging after treatment with nano-fractional RF. The authors reported the improvement of photodamage, pigmentation, and wrinkles in all cases; however, a significant improvement of erythema, dryness, edema, crusting, and healing due to RF procedure was observed after 24 hours of procedure in the subjects who took the gel mask. 

Striae distensae is observed as destruction of elastic and collagen fibers in pathological view²⁹ in which the effectiveness of carboxytherapy has been reported. 

 Podgórna et al²⁹ studied the efficacy of carboxytherapy in treating striae distensae through investigation of cutometric parameters in 15 women after three sessions of treatment at one-week intervals. They showed a significant effectiveness of this modality in improvement of skin elasticity. The analysis of photographic views revealed 58 percent improvement of striae distensae visibility with decreased width and length of the lesions and change of the color lesions to natural skin one.²⁹ 

In a split-body trial that included 20 participants, Hodeib et al³⁰ assessed the efficacy of carboxytherapy in comparison with platelet-rich plasma (PRP) in treatment of striae albae. PRP injection and carboxytherapy were performed on the right and left sides of the abdomen, respectively, every 3 to 4 weeks for four sessions. Based upon clinical examination and patient satisfaction, a significant improvement was reported with both therapies, with no significant difference between them. IHC examination significantly demonstrated high expression of fibronectin in both treatments with significantly higher in carboxytherapy group. 

El-Domyati et al¹⁷ assessed the efficacy of carboxytherapy in the treatment of striae distensae in clinical and pathological examinations. The clinical assessment showed statistically significant improvement, and based upon histometric analysis, a statistically significant increase in the epidermal thickness was seen. Histochemical examination of elastic and collagen fibers showed increased content and density of collagens, organized in bundles and parallel to the epidermis, and better structure of curled and fragmented elastic fibers.

Farouk et al³¹ evaluated the efficacy of carboxytherapy in the treatment of striae distensae in 30 adult female participants. The therapy was done once a week for six sessions. Their findings showed the effectiveness and safety of carboxytherapy in management of this skin condition.

In our study, assessment of hematoxylin-eosin-stained slides revealed the statistically significant effect of carboxytherapy in increasing the thickness of the epidermis and dermis. As thinness of the epidermis and dermis, and less prominence of the rete ridges are pathological manifestations of skin aging^{4, 5} increase of the epidermal thickness by carboxytherapy confirmed the effectiveness of this procedure in controlling and reversing the aging manifestations. 

Masson-trichrome staining showed significantly increased production of collagen in our study. As decreased production of the type I collagen was seen in intrinsically aged skin due to downregulation of the TGF-β/Smad signaling⁹, increase of this type of collagen in our study confirmed the effectiveness of carboxytherapy in the management and in reversing skin aging, partially through increasing the type I collagen seemingly through upregulation of TGF-β/Smad signaling. The latter was confirmed through increased expression of TGF-β in the IHC examination of this factor in our study.

Regarding the effect of anti-aging modalities on the elastin content in the ECM, studies have shown controversial results. In our study, staining with Orcein Giemsa showed a statistically significant increase in the synthesis of elastin. As a progressive loss of elastic fibers is seen in the intrinsic skin aging process,⁷ increased production by carboxytherapy confirmed the effectiveness of this modality in managing and reversing the skin aging process in our study. 

As considerable impairment of cutaneous vascular networks is a pathologic manifestation of skin aging which causes reduction of microvasculature,²⁴ increasing VEGF as an important angiogenic growth factor can manage, and even reverse the aging process. In our study, a significant increase of VEGF expression levels in the IHC examination confirmed the effectiveness of carboxytherapy in treating and reversing the skin aging partially through promotion of angiogenesis. 

A low number of subjects, and a restricted number of studied variables in IHC examination necessitate a robust study with more cases and consideration of more variables in IHC examination for precise confirmation of the effectiveness of carboxytherapy in treating skin aging. 

Conclusion

Skin aging as a complex process is defined an inevitable physiological process which results in fine wrinkles, xerosis, progressive dermal atrophy, loss of skin elasticity, and skin laxity.⁹ In previous studies, the effectiveness of carboxytherapy as a safe, minimally invasive modality has been shown in skin rejuvenation, restoration, and recondition.¹¹ In the current study, we evaluated the effectiveness of this modality in the treatment of skin aging through pathological and IHC examinations.

Acknowledgments

The authors thank Mr. Iman Farahani, executive manager of the specialty clinics of Valiasr Hospital of Arak, Iran, Mrs. Maryam Joodaki, nurse of the specialty clinics of Valiasr Hospital of Arak, Iran, Mrs. Latifeh Faridi, and Mrs. Zahra Zarei for supporting us in recruiting participants.   

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