|Year : 2021 | Volume
| Issue : 1 | Page : 35-40
Clinical Significance of Origin of Cystic Artery and its Relations to Calot's Triangle
Swapna Thampi1, Renuka Krishnapillai2, Revathi Murugesan3, Suja Robert Sarasammal1, Umesan Kannan vilakom Govindapillai1
1 Associate Professor, Department of Anatomy, Government Medical College, Trivandrum, Kerala, India
2 Professor and HOD, Department of Anatomy, Government Medical College, Trivandrum, Kerala, India
3 Senior Resident, Department of Anatomy, Government Medical College, Trivandrum, Kerala, India
|Date of Submission||27-Sep-2020|
|Date of Decision||11-Dec-2020|
|Date of Acceptance||22-Dec-2020|
|Date of Web Publication||27-Jan-2021|
Suja Robert Sarasammal
Department of Anatomy, Government Medical College, Trivandrum- 695011, Kerala
Source of Support: None, Conflict of Interest: None
Background and Aim: Cystic artery (CA) is one of the important structures to be ligated during cholecystectomy. Recurring complications such as hemorrhage or liver injury has attracted surgeons, radiologists, and anatomists to do research on the topic. Materials and Methods: Thirty-two formalized cadavers were used for this study. After opening the abdomen, lesser omentum was separated, followed by fine dissection of CA. Findings were recorded and variations were photographed. The collected data were analyzed, and the prevalence was expressed as percentage. Results: In 68.75%, single CA was passing through the Calot's triangle (CT), and in 4.5%, double CA was passing through the CT. Conclusion: Accurate knowledge of CA anatomy is essential to avoid iatrogenic extrahepatic biliary injuries in surgeries related to this region. We have focused to explore CA variations to help the surgeons and radiologists.
Keywords: Calot's triangle, cholecystectomy, cystic artery
|How to cite this article:|
Thampi S, Krishnapillai R, Murugesan R, Sarasammal SR, vilakom Govindapillai UK. Clinical Significance of Origin of Cystic Artery and its Relations to Calot's Triangle. Natl J Clin Anat 2021;10:35-40
|How to cite this URL:|
Thampi S, Krishnapillai R, Murugesan R, Sarasammal SR, vilakom Govindapillai UK. Clinical Significance of Origin of Cystic Artery and its Relations to Calot's Triangle. Natl J Clin Anat [serial online] 2021 [cited 2022 Jan 23];10:35-40. Available from: http://www.njca.info/text.asp?2021/10/1/35/308118
| Introduction|| |
Cholelithiasis is often treated with laparoscopic cholecystectomy.,, During the procedure, cystic artery (CA) is ligated and later cystic duct is ligated close to the common bile duct (CBD). If an uncontrolled bleeding is encountered (reported incidence: 0%–0.9%), the laparoscopic procedure may be converted into open cholecystectomy.,, Therefore, a thorough knowledge of CA is essential for decreasing complications. CA originates frequently from right hepatic artery (RHA). Later, it divides into superficial and deep branches and gives off numerous twigs to the liver., Michels noted double CA in 25% of participants, where the superficial and deep branches of the CA originated separately. Calot's triangle (CT) was first described by Jean-Francois Calot (1861–1944). The triangle was an isosceles triangle. The superior and inferior sides represented by the CA and cystic duct are equal and little longer than the part of the hepatic duct (medially). In modern definition of CT, it is bounded superiorly by the inferior surface of the liver and inferiorly by the cystic duct and medially by the common hepatic duct (CHD)., Hence, the CT is also sometimes referred to as cystohepatic or hepatocystic triangle. This modern triangle provides a constant boundary to the surgeon. The CA along with the RHA, cystic lymph node of Lund, lymphatics, and connective tissue constitutes the contents of CT. Of the various structural injuries following cholecystectomy, bile duct injury is most feared. Among vascular injuries, the most common structure injured during laparoscopic cholecystectomy is RHA. It may be mistaken for CA and can be clipped. Accidental ligation of RHA occurs in cases where short CA arises from Moynihan's hump of RHA in CT. In this condition, injury to RHA leads to liver necrosis.,, Vascular injuries are almost always encountered in association with bile duct injuries. Structural injuries can be prevented using intraoperative cholangiography. Hence, clipping or division should not be done without dissecting CT clearly and identifying CA and cystic duct conclusively. Gupta and Jain have stated that the CA should be divided on the right side of lymph node of Lund close to the gallbladder to avoid injury to the RHA.
A replaced RHA travels close to the cystic duct and gallbladder giving numerous branches. On laparoscopic examination, the replaced hepatic artery appears to be a large CA, accidental ligation of which can cause liver hypoxia. The CA arising from the left hepatic artery or gastroduodenal artery (GDA) cannot be visualized within CT during laparoscopic cholecystectomy. When CA travels inferior to the cystic duct, it becomes the first structure met during cholecystectomy. When CA is anterior to CHD, there is danger of bile spillage. For clear visualization of CA in CT, traction can be applied to fundus of the gallbladder over liver margin with lateral traction applied to the infundibulum of the gallbladder. Numerous variations in origin and course of CA can cause complications in cholecystectomy. Awareness of these variations is a prerequisite for meticulous dissection. The present study aims to document the origin and course of normal CA. We also aim to study the variations in origin of CA, its course, and relations with respect to CT in detail. The results will be compared to the existing literatures and analyzed. We expect this study to further add to existing knowledge which, in turn, may help the surgeons to anticipate procedure-related morbidities.
| Materials and Methods|| |
This was a descriptive cross-sectional study conducted in 32 formalin-fixed cadavers over a period of 3 years from 2016 to 2019 after receiving permission from the Institutional Ethics Committee (IECNO.02/26/2018LMCT dated January 19, 2018). After opening the abdomen, lesser omentum was separated, followed by fine dissection of CA. The course of CA with its relation to CT was recorded. The relation of CA to cystic duct and biliary ducts was also noted. Findings were recorded, and variations were photographed. The collected data were analyzed, and the prevalence was expressed as percentage.
| Results|| |
CA variations in relation to CT have been classified into three groups in accordance with the classification used in a study by Ding et al.
In this type, the CA passes through the CT, which was seen in 22 cases (68.75%). This includes two subgroups: classical single CA seen in 21 cases (95.45%) and double CA seen in one case (4.5%).
Single CA here originated from the RHA in 61.9%, middle hepatic artery (MHA) in 19% [Figure 1], and aberrant RHA (ABRHA) arising from the superior mesenteric artery (SMA) in 6 instances (19%). The ABRHA after arising from SMA runs upward behind GDA and portal vein, anterior to inferior vena cava, posterior to CD to enter the CT, and gives short CA that enters the neck of the gallbladder. In two cases, the RHA was coursing behind CHD producing a sinuosity within the CT and was very close to the gallbladder and cystic duct. It is known as Moynihan's hump, which gave very short cystic arteries [Figure 2]. In two cases, the CA after arising from the RHA was seen to pass through a ductal sling. The ductal sling was suspended from the right hepatic duct in one case, and it communicated between the CHD and the right hepatic duct in another [Figure 3]. A rare case was seen where CA originated from the ABRHA (from SMA) within the CT and immediately divided into superficial and deep. The deep branch continued through the gallbladder bed, while the superficial branch received a communication from MHA which was arising from the left hepatic artery [Figure 4]. Double CA was seen in 4.5% of cases where the origin of superficial and deep branches was taking place independently from RHA [Figure 5].
|Figure 1: Cystic artery originating from the middle hepatic artery. Cystic artery divides into superficial and deep branches (reaches gallbladder bed). Left hepatic artery also seen|
Click here to view
|Figure 2: Right hepatic artery forming Moynihan's hump within Calot's triangle from which short cystic artery arises. Cystic duct and common bile duct (GB- Gall bladder, CA- Cystic artery, RHA- Right hepatic artery, CD- Cystic duct, CBD- Common bile duct)|
Click here to view
|Figure 3: Cystic artery originating from the right hepatic artery high up in Calot's triangle since the origin is being suspended by a ductal sling suspended from the right hepatic duct (GB- Gall bladder, CA- Cystic artery, RHA- Right hepatic artery, CD- Cystic duct, CBD- Common bile duct, CHD- Common hepatic duct)|
Click here to view
|Figure 4: Aberrant right hepatic artery (ABRHA) entering the Calot's triangle (CT) and giving cystic artery (CA) which divides into superficial (coronary arteries) and deep (coronary artery disease) branch. Middle hepatic artery (MHA) arising from the left hepatic artery and gives a branch which passes anterior to common hepatic duct (CHD) to join with the coronary arteries|
Click here to view
|Figure 5: Double cystic artery (CA) arising from right hepatic artery (RHA) while passing behind the common hepatic duct (CHD) and crossing Calot's triangle (CT)|
Click here to view
This represents outside CT type, which means that the CA cannot be seen in the CT and was seen in 9 cases (28.13%). In one case, CA originated from RHA, and it passed anterior to the CHD, in anterior aspect of the CT, and entered through the neck of the gallbladder. In three cases, CA originated from proper hepatic artery (PHA) and passed anterior to CHD toward the neck of the gallbladder. In one case, superior pancreaticoduodenal (SPD) artery gave CA immediately after originating from SMA. The CA then ran obliquely upward behind CBD along the lower border of the cystic duct to enter the neck of the gallbladder [Figure 6]. In three cases where CA arouses from the ABRHA which was originating from GDA in two cases [Figure 7] and aorta in one case [Figure 8]. In one case, RHA passes posterior to CHD, traverses CT, and passes deep in the groove for the ligamentum teres from where CA originates and enters through the gallbladder bed to continue as deep branch. No superficial branch was seen.
|Figure 6: The cystic artery arises from superior pancreaticoduodenal artery courses upward behind the common bile duct and remains outside the Calot's triangle inferior to the cystic duct|
Click here to view
|Figure 7: Cystic artery originates from the aberrant hepatic artery which arises from the gastroduodenal artery. Cystic artery runs anterior to the common hepatic duct and cystic duct|
Click here to view
|Figure 8: Cystic artery is originating from the aberrant right hepatic artery which arises from aorta|
Click here to view
Thirteen percent (four cases) belonged to this group gallbladder has more than one blood supply. CA existed both inside and outside the CT, which has been named as compound CA type. Here, one CA originated from PHA, traversed anterior to CT, and continued as superficial branch. The other CA was arising from RHA within the CT and continued as the deep branch.
Cystic artery variations as per source of origin and number
In our study, single CA was seen in 30 cases (93.75%) and double CA was seen in 2 cases (6.25%). The various origins of CA were from RHA (51.72%), ABRHA (21.87%), MHA (13.79%), PHA (10.34%), and SPD (3.44%). Among double CA, both the arteries came from RHA.
Branching pattern of cystic artery
In 84.37%, the CA divided into superficial and deep branches. In 15.63% of cases, only a single branch existed. Out of these in three cases, CA continued as superficial branch, and in two cases, it continued as deep branch.
Relation of cystic artery to common hepatic duct and common bile duct
CA coursed posterior to CHD in 57.6% and anteriorly in 21.21%. CA passed anterior to CBD in 3% and posteriorly in 87.9% when CA was arising from SPD artery. In 9.1%, CA had no relation with CBD or CHD when it originated from caterpillar hump within CT and where CA was arising from RHA deep within the groove for the ligamentum teres. When CA was arising from SPD artery, it passed below the cystic duct [Figure 6].
| Discussion|| |
CA displays diverse variations in and around CT,, and in this study, the prevalence was 37.5% of cases. Therefore, careful dissection of CT is decisive for cholecystectomy. The CA was seen within the CT in 62.5% of cases in this study compared to 85.5% of cases in a study by Ding et al. The CA was found within CT in studies of Aristotle (92.5%), Zubair et al. (75.4%), Balija et al. (73.5%), and Suzuki et al. (76.6%). The position of CA was variable in CT. CA passing through the middle of CT was of higher proportion (39%) similar to a study by Dandekar and Dandekar (47.6%). CA outside the CT was seen in 28.13% in the present study as compared to 13% in a study by Ding et al. CA from GDA was seen in 7.5% in their study, while it was absent in the present study. CA from ABRHA was seen in 3%, while it was 33.33% in the present study. CA from the liver parenchyma was 2.5%, but we had no such cases. Instead, in 11.11% of cases, CA originated from a RHA which was seen deep in the groove for the ligamentum teres. CA originated from the left HA in 1% of cases in their study, while we did not have any such case. In our study, there was CA arising from RHA (11.11%), PHA (33.33%), and SPD (11.11%), all of which traversed outside CT, while no such cases were seen in a study by Ding et al. CA originating outside CT becomes the first structure to be confronted during exploration of CT. The CA both inside and outside the CT type was reported as 1.5% of cases by Ding et al., while the present study had 9.1% of cases, which is similar to the reports by Zubair et al. (5.46%) and Suzuki et al. (7.37%).
CA originated from RHA was 51.72% as compared to other studies by Dandekar and Dandekar (79.3%), Suzuki et al. (79.09%), Balija et al. (94.5%), Tejaswi et al. (92%), and Pushpalatha and Shamasundar (54%). In a study of CA variation review by Andall et al. in 9836 cases, in 79.02%, CA originated from RHA, in 0.34% of cases, the CA was absent, and in 8.9% of cases, there were more than one CA present. In our study, in 15.4% of cases, the RHA was coursing behind the CHD before forming a single loop of caterpillar hump (Moynihan's hump). Studies reporting its incidence are Benson and Page (5–15%), Bergamaschi and Ignjatovic (12.9%), and Carol et al. (6–16%). The CA arising from caterpillar hump is short and may get avulsed from RHA during gallbladder removal. Giant CA in CT may suggest the presence of caterpillar hump. One more variation, CA arising from RHA and then passing through a ductal sling is seen in 11.1%. Another rare case seen in the present study was CA after arising from an ABRHA (from SMA) divided into superficial and deep branches. The superficial branch received a communicating branch from MHA which passed anterior to CHD. CA also arose from MHA in 13.7%, PHA 10.34%, and ABRHA in 33.3%. Such case was not reported in other studies.
CA arising from MHA is rare. In our study, in 13.79% of cases, the CA arose from MHA, which is consistent with a study by Bharadwaj. CA originating from LHA was reported by Balija et al. in 1% of cases and by Ramakrishna and Tiwari in 2% of cases but was not seen in our study. During laparoscopic exploration, a single large CA needs care since it might be an aberrant hepatic artery. Commonly, the ABRHA arises from SMA., In this study, in 18.75% of cases, the CA arose from ABRHA, out of which 50% originated from SMA, 33.3% from GDA, and 16.7% from aorta. During cholecystectomy, ligation of ABRHA has been linked to liver ischemia, necessitating resection of affected lobe. In the present study, double CA was seen in 10% of cases, which is similar to the study done by Ding et al. The frequency of double CA varies from 15% to 25%. Deep branch of CA if absent may indicate the presence of double CA. The prevalence reported by different authors is shown in [Table 1]. An accessory CA is susceptible to injury if left unidentified. The superficial branch of double CA originated from RHA, LHA, MHA, and GDA, and deep branch came from RHA. The superficial branch arose from RHA, ABRHA, PHA, CHA, or LHA, while the deep branch arouse from RHA, ABRHA, PHA, or GDA. In their study, 18.3% of cases showed origin of both superficial and deep branches of double CA from RHA. The same pattern was observed in 3.12% of cases in our study. In case of double CA, accidental ligation of one of the vessels originating far from the bile duct may complicate the surgery. CA variations have been explained by selective degeneration of different abdominal aortic vessels and its branches supplying the hepatic diverticulum. In this study, the CA coursed posterior to CHD in 57.6% and anteriorly in 21.21%. CA passed anterior to CBD in 3% and posteriorly in 87.9% when CA was arising from SPD artery. In 9.1%, CA had no relation with CBD or CHD. Rahman and Anwars reported that the CA crossed anterior to CHD in 6.67%, posterior to the CHD in 90% and in 3.3% the CA passed below the cystic duct.. In our study in one case where CA arose from SPD artery, it passed below CD.
|Table 1: Tabulation of various parameters of cystic artery from previous studies|
Click here to view
Aristotle reported that CA crossed anterior to CHD in 10% and posterior to RHD in 90%. CBD or CHD may get injured when it is being crossed anteriorly by CA. Various parameters from previous studies are summarized in [Table 1]. The incidence of structural injury and new techniques in surgery for enhancing the visualization of CT are reviewed in a study by Abdalla et al.
| Conclusion|| |
CA is the main structure to be isolated and ligated during cholecystectomies and hence variations have to be kept in mind. This will help to avoid hemorrhage or hepatobiliary complications.
The authors thank all the body donors, who donated their bodies for academic and research purposes, and all the faculties who supported in different steps of research.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Tebala GD. Three port laparoscopic cholesystectomy by harmonic dissection without cystic duct and artery clipping. Am J Surg 2006;191:718-20.
Kamitani S, Tsutamoto Y, Hanasawa K, Tani T. Laparoscopic cholesystectomy in situ
inversus totalis with inferior cystic artery: A case report. World J Gastroenterolgy 2005;11:5232-4.
Huscher CG, Lirici MM, Dipaola M, Crafa F, Napolitano C, Mereu A, et al
. Laparoscopic cholesystectomy by ultrasonic dissection without cystic duct and artery ligature. Surg Endosc 2003;17:442-51.
Mcgregor LE. Synopsis of Surgical Anatomy. 12th
ed. Bristol K.M. Varghese Company; 1986.
Ding YM, Wang B, Wang WX, Wang P, Yan JS. New classification of the anatomic variations of cystic artery during laparoscopic cholesystectomy. World J Gastroenterol 2007;13:5629-34.
Nagral S. Anatomy relevant to cholecystectomy. J Minim Access Surg 2005;1:53-8.
Vishnumaya G, Potu BK, Gorantia VR. Anomalous origin of cystic artery from gastroduodenal artery a case report. In J Morphol 2008;26:75-6.
Standring S. In: Standring S, editor. Gray's Anatomy: The Anatomical Basis of Clinical Practise. 41st
ed. London: Elsevier; 2016. p. 1176.
Hollinshead WH. Anatomy for Surgeons: The Thorax, Abdomen and Pelvis. 2nd
ed. New York, USA: Harper and Row; 1971.
Michels NA. The hepatic, cystic and retroduodenal arteries and their relations to the biliary duct: With samples of entire celiacal blood supply. Ann Surg 1951;133:503-24.
Calot JF, Cimmino PT, Bocchetti T, Izzo L. Anatomo-surgical considerations in laparoscopic chole cystectomy. G Chir 1992;13:149-51.
Haubrich WS. Calot of the triangle of Calot. Gastroenterology 2002;123:1440.
Mrksic MB, Farkas E, Cabafi Z, Komlos A, Sarac M. Complications in laparoscopic cholecystectomy. Med Pregl 1999;52:253-7.
Alves A, Farges O, Nicolet J, Watrin T, Sauvanet A, Belghiti J. Incidence and consequence of an hepatic artery injury in patients with postcholecystectomy bile duct strictures. Ann Surg 2003;238:93-6.
Cuschieri A. Disorders of the biliary tract. In: Cuschieri A, Steele RJ, Moossa AR, editors. Essential Surgical Practice. 4th
ed. London: Arnold; 2002. p. 375-452.
Stewart L, Robinson TN, Lee CM, Liu K, Whang K, Way LW. Right hepatic artery injury associated with laparoscopic bile duct injury: Incidence, mechanism of consequences. J Gastrointest Surg 2004;8:523-31.
Shayani V, Jacobs L, Sackier J. Laparoscopic cholecystectomy II. In: Kavic MS, Levinson CJ, editors. Prevention and Management of Laparoscopic Surgical complications. Miami: Society of Laparoendoscopic Surgeons; 1999.
Strasberg SM, Herti M, Soper NJ. An analysis of the problem of billiary injury during laparoscopic cholecystectomy. J Am Coll Surg 1995;180:101-25.
Gupta V, Jain G. Safe laparoscopic cholecystectomy: Adoption of universal culture of safety in cholecystectomy. World J Gastrointest Surg 2019;11:62-84.
Cimmino PT, Bocchetti T, Izzo L. Anatomo-surgical considerations in laparoscopic cholecystectomy. G Chir 1992;13:149-51.
Larobina M, Nottle PD. Extrahepatic biliary anatomy at laparoscopic cholesystectomy: Is aberrant anatomy important? ANZ J Surg 2005;75:392-5.
Aristotle S. Variations in origin and course of cystic artery and its relations to Calot's triangle with its clinical implications. OA Anat 2014;2:17.
Zubair M, Habib L, Mirza MR, Cnanna MA, Yousuf M, Quraishy MS. Anatomical variations of cystic artery telescopic facts. Med J Malaysia 2012;67:494-6.
Balija M, Huis M, Nikolic V, Stulhofer M. Laparoscopic visualisation of cystic artery anatomy. World J Surgery 1999;23:703-7.
Suzuki M, Akaishi S, Rikiyama T, Naitoh T, Rahman MM, Matsuno S. Laparoscopic cholesystectomy, Calot's triangle, and variations in cystic arterial supply. Surg Endosc 2000;14:141-4.
Dandekar U, Dandekar K. Cystic Artery: Morphological Study and Surgical Significance. Anat Res Int. 2016;2016:7201858. doi: 10.1155/2016/7201858. Epub 2016 Oct 16. PMID: 27822387; PMCID: PMC5086348.
Hugh TB, Kelly MD. Laparoscopic anatomy of cystic artery. Am J Surg 1992;163:593-5.
Tejaswi HL, Dakshayani KR, Ajay N. Prevalence of anatomical variations of cystic artery in South Indian cadavers. Int J Res Med Sci 2013;1:424-8.
Pushpalatha K, Shamasundar NM. Variation in the origin of cystic artery. J Anat Soc India 2010;59:35-7.
Andall RG, Matusz P, du Plessis M, Ward R, Tubbs RS, Loukas M. The clinical anatomy of cystic artery variations: A review of over 9800 cases. Surg Radiol Anat 2016;38:529-39.
Benson EA, Page RE. A practical reappraisal of the anatomy of the extrahepatic bile ducts and arteries. Br J Surg 1976;63:854.
Bergamaschi R, Ignjatovic D. More than two structures in Calot's triangle: A Postmortem study. Surg Endosc 1999;14:354-7.
Carol EH, Conner S, David LD. Operative Anatomy. 3rd
ed. Philadelphia, PA: Lippincott, Williams and Wilkins; 2008.
Schwartz SI. Maingot's Abdominal Operations: Anatomy of the Extrahepatic Biliary Tract. 3rd
ed., Vol. 2. London, UK: Prentice Hall International, Appleton and Lange; 1990.
Conner CE, Hall TJ. Variant arterial anatomy in laparoscopic cholesystectomy. J Minim Access Surg 1992;1:590-2.
Bharadwaj N. Anomalous origin of hepatic artery and its significance for hepatobiliary surgery. J Anat Soc India 2010;59:173-6.
Ramakrishna R, Tiwari S. Cystic artery: An anatomic morphological study and its clinical significance. Natl J Clin Anat 2019;8:10-3. [Full text]
Jones RM, Hardy KJ, The hepatic artery: A reminder of surgical Anatomy. J R Coll Surg Edinb 2001;46:168-70.
Hiatt JR, Gabbay J, Busuttil RW. Surgical anatomy of the hepatic arteries in 1000 cases. Ann Surg 1994;220:50-2.
Uenishi T, HirohashiI K, Tanaka AN, Fujio N, Kubo S, Kinoshita H. Right hepatic lobectomy for recurrent cholangitis after bile duct and hepatic artery injury during laparoscopic cholecystectomy: Report of a case. Hepatogastroenterology 1999;46:2296-8.
Daseler EH, Anson BJ, Hambley CW, Reiman AF. The cystic artery and the constituents of hepatic pedicle. Surg Gyneacol Obstet 1947;85:47-63.
Saidi H, Karnaja TM, Ogengo JA. Variant anatomy of the cystic artery in adult Kenyans. Clin Anat 2007;20:943-5.
Johnston EV, Anson BJ. Variations in the formation and vascular relationships of the bile ducts. Surg Gynaecol Obstet 1947;85:47-63.
Bakheit M. Prevalence of the variation of cystic artery in Sudanese. Rev Sant Mediterr Orient 2009;15:1308-12.
Browne EZ. Variations in origin and course of the hepatic artery and its branches. Surgery 1940;8:424-45.
Nowak M. Variations of the cystic artery in man. Folia Morphol 1977;36:89-98.
Rahman KM, Anwars S. Presence of cystic artery in the Calot's triangle and its relation with common hepatic duct-a postmortem study. Bangladesh J Anat 2012;10:50-6.
Abdalla S, Pierre S, Ellis H. Calot's triangle. Clin Anat 2013;26:493-501.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]