Analysis of glycoconjugates and

morphological characterization of the

descending colon and rectum of the
plains viscacha, Lagostomus maximus
Mara Florencia Tano De La Hoz & Mirta
Alicia Flamini & Enrique Leo Portiansky
& Alcira Ofelia Daz
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Accepted Manuscript

Title: Analysis of glycoconjugates and morphological

characterization of the descending colon and rectum of the
plains viscacha, Lagostomus maximus

Authors: Marı́a Florencia Tano de la Hoz, Mirta Alicia

Flamini, Enrique Leo Portiansky, Alcira Ofelia Dı́az

PII: S0944-2006(18)30240-X
DOI: https://doi.org/10.1016/j.zool.2019.06.001
Reference: ZOOL 25691

To appear in:

Received date: 21 December 2018

Revised date: 29 May 2019
Accepted date: 3 June 2019

Please cite this article as: de la Hoz MFT, Flamini MA, Portiansky EL, Dı́az AO,
Analysis of glycoconjugates and morphological characterization of the descending
colon and rectum of the plains viscacha, Lagostomus maximus, Zoology (2019),
https://doi.org/10.1016/j.zool.2019.06.001

This is a PDF file of an unedited manuscript that has been accepted for publication.
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apply to the journal pertain.
Analysis of glycoconjugates and morphological characterization of the descending

colon and rectum of the plains viscacha, Lagostomus maximus

Short title: Descending colon and rectum of L. maximus.

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María Florencia Tano de la Hoz a,d,*, Mirta Alicia Flamini b, Enrique Leo Portiansky c,d, and

Alcira Ofelia Díaz a

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a N
Instituto de Investigaciones Marinas y Costeras (IIMyC), Departamento de Biología,
A
FCEyN, CONICET-Universidad Nacional de Mar del Plata, Funes 3250 (7600), Mar del
M

Plata, Argentina.
b
Laboratorio de Histología y Embriología Descriptiva, Experimental y Comparada,
D

Departamento de Ciencias Básicas, Facultad de Ciencias Veterinarias, 60 y 118 (1900),

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Universidad Nacional de La Plata, La Plata, Argentina.

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c
Laboratorio de Análisis de Imágenes, Facultad de Ciencias Veterinarias, Universidad

Nacional de La Plata (LAI, FCV-UNLP), 60 y 118 (1900), La Plata, Argentina.

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d
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
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*
Corresponding author: M.F. Tano de la Hoz, IIMyC, FCEyN, CONICET-Universidad

Nacional de Mar del Plata. Funes 3250 3° piso, 7600 Mar del Plata, Buenos Aires,

Argentina. E-mail: mftano@mdp.edu.ar

1
Graphical abstract

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Comparative analysis of the morphology, ultrastructure and glycosylation pattern of the

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descending colon and rectum of Lagostomus maximus. Histochemical results (HQ) revealed

that in both sectors of the large intestine, there are goblet cells (GC) with different

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glycosylation pattern (GCa, GCb and GCc) within a morphologically homogeneous cell
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population. Specific differences between both intestinal segments were revealed by lectin
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histochemistry (LHQ). Arrow, glycocalyx; *, lumen. Scale bar: 300 µm (B); 20 µm (A, C).
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D
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Highlights

 Goblet cells form a homogeneous cell population at ultrastructural level.

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 The glycosylation pattern of goblet cells depends on the intestinal region studied.


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Sulphates are the main group responsible in determining the acid gradient of mucus.

 The glycocalyx of both anatomical regions showed carboxylated glycoconjugates.

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2
Abstract

Herbivores exhibit specializations at the intestinal level that facilitate the bacterial

fermentation. The available information on the digestive physiology of Lagostomus

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maximus makes this rodent an interesting model to evaluate morpho-functional adaptations

to herbivory. The general objective of this work was centered on the study of the

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morphology and histochemistry of the descending colon and rectum of L. maximus. To do

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so, a comparative analysis of the morphology, ultrastructure and glycosylation pattern of

both anatomical regions was carried out. Histochemical results revealed that in both sectors

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of the large intestine, there are goblet cells with different glycosylation pattern within a

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morphologically homogeneous cell population. The main difference between both intestinal
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segments lay in the fact that the most distal region of the large intestine showed a greater
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proportion of sialomucins, characterized by being slightly O-acetylated. Further specific

differences were revealed by lectin histochemistry. These data allowed to perform a

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functional interpretation of the cell types and secreted substances, thus contributing to a
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better understanding of the role of mucins in the intestinal tract functioning.

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Key words: Hystricognathi, large intestine, mucins, histochemistry, morphology.

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3
1. Introduction

Herbivores depend on microbial fermentation to use the cellulose as their main source of

energy. Many herbivorous vertebrate species possess specialized regions in the digestive

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tract, such as fermenting chambers, where symbiotic microorganisms degrade cellulose

(Kardong, 2015). Whenever digestion of homopolysaccharides is carried out in a

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specialized stomach, it is said to be a gastric fermentation, while the microbial digestion

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centered in the intestine is called intestinal fermentation (Sakaguchi, 2003; Kardong, 2015).

A low bacterial density is present in the intestinal fermenters of the small intestine in

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comparison to the large intestine due to the efficient trapping of bacteria by mucus and its

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rapid transport to the distal portion of the intestinal tract (Hansson, 2012).
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The mucus forms a highly hydrated gel over the intestinal mucosa. It is mainly
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composed by mucins, and other minor components such as salts, immunoglobulins and

growth factors. Mucins are high molecular weight glycoproteins with a high content of O-
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linked oligosaccharides and a smaller proportion of N-linked oligosaccharides. Their main

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functions in the large intestine are to limit the contact of bacteria with the epithelium and to
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facilitate their transport toward the distal region of the intestinal tract (Robbe et al., 2004;

Kim and Ho, 2010).

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Comparative studies in diverse vertebrate species have demonstrated that the

morphology of the digestive tract varies significantly, even among related groups, by
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exhibiting structural modifications adapted to specialized diets (Kardong, 2015). In

particular, rodents present noticeable differences in their intestinal anatomy, mainly in the

caecum and colon (Kotzé et al., 2010).

4
 Plains viscachas, Lagostomus maximus, are Hystricognathi (Rodentia, Caviomorpha) in

the Chinchillidae family, that inhabit Argentina, Bolivia and Paraguay. They are

exclusively herbivore rodents that in their natural environment feed on a great variety of

plant species, showing preference for grasses and dicotyledons (Puig et al., 1998). The

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structural organization of the intestinal tract of L. maximus is similar to that described in

other hystricognath rodents, by presenting a voluminous caecum and a particularly long

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colon (Clauss et al., 2007; Hagen et al., 2015). Moreover, using markers for digestibility

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essays it was shown that L. maximus practices caecotrophy and presents a colonic groove

along the mesenteric side of the ascending colon (Clauss et al., 2007; Hagen et al., 2015).

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In this way, this hystricomorph rodent exhibits, as other herbivores, specializations at the

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intestinal level that facilitate the bacterial fermentation. The available information on the
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digestive physiology of L. maximus makes this rodent an interesting model to evaluate
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morpho-functional adaptations to herbivory.

In the last years, our research group has studied the histochemical profile of the goblet
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cells along the small intestine of L. maximus (Tano de la Hoz et al., 2014; 2016). Also, we
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have recently demonstrated that the glycosylation pattern of mucus has a key role in the
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functioning of the colonic groove of L. maximus (Tano de la Hoz et al., 2017). To further

studying the large intestine, the main goal of this work was centered on the morphology and
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histochemistry of the descending colon and rectum of L. maximus. Thus, a comparative

analysis of the morphology, ultrastructure and glycosylation pattern of both anatomical

A

regions was carried out.

2. Materials and methods

2.1 Animals

5
 Wild adult plains viscachas, Lagostomus maximus, Desmarest, 1817 of both sexes (n=

14; 8 females and 6 males) from the Estación de Cría de Animales Silvestres (ECAS),

Ministry of Agroindustry of the Province of Buenos Aires (Argentina) were used.

Captures were made during the periods of March-April, July-August and December-

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January, years 2009-2014. Captured animals were anesthetized with xylazine (8 mg/kg

body weight), followed by ketamine (50 mg/kg body weight) by intramuscular via

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(Ketanest, Laboratorio Scott Cassara). Each anesthetized animal was weighed and sexed by

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the traditional anus-genital distance method. Average body weights of females were 4-5.5

kg, while males weighed between 7-8.5 kg. Sanitary status of animals was evaluated,

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especially observing the hairy cover condition and the presence of ectoparasites and

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tegumentary injuries. Only healthy animals were used for this study. Once they reached the
A
deep plane of anesthesia, intracardiac perfusion was conducted using physiological saline
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solution, followed by paraformaldehyde 4% in phosphate buffer 0.1M. The method used

was approved by the Institutional Committee for the Caring and Use of Laboratory Animals
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(CICUAL) of the School of Veterinary Sciences, National University of La Plata (52-4-

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15T) and is in accordance with the international recommendations for the use of
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experimental animals (National Research Council, 2011).

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2.2 Sampling and histological processing

Necropsies were carried out immediately after euthanasia. For histological and
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histochemical analyses, three samples from the cranial portion of the descending colon and

rectum were taken per animal. Samples were processed for inclusion in paraffin and 4µm

thick sections were stained with hematoxylin-eosin (H-E) and Masson’s trichrome. From

each sample, at least forty-two serial cross sections were obtained. Each technique was
6
repeated in duplicates for each sample. Images were taken using a trinocular microscope

(CH30, Olympus, Japan).

2.3. Ultrastructural study

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To perform an ultrastructural study of the intestinal epithelium, 0.5-1 mm3 samples from

the descending colon and rectum of L. maximus were taken. Sections were fixed in cold

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(4°C) 2% glutaraldehyde in 0.1 M phosphate buffer solution, pH 7.3, at 4°C during 2 h.

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Afterwards, the samples were exposed to three folds washings with 0.1 M phosphate-

buffered saline (PBS), pH 7.2, during 20 min each. Samples were then post fixed in 1%

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osmium tetroxide at 4°C during 1 h, dehydrated in ethanol solution at increasing
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concentrations and included in Epon 812. To select the area of study semi thin cuts were
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stained with toluidine blue for optical microscope observation. Then, ultra-fine cuts were
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contrasted with uranyl acetate and 1% plumb citrate. Samples were examined using a JEOL
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JEM 1200EX II electronic transmission microscope (TEM) and photographed with a digital
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camera (ES 500W Erlangshen Gatan CCD).

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2.4 Histochemical study for the determination of glycoconjugates

The histological sections were also exposed to the following histochemical techniques to
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characterize glycoconjugates (GCs) and to evaluate their distribution and possible

A

physiological roles:

2.4.1 Periodic acid-Schiff’s reagent (PAS). It is used to evidence GCs with oxidizable

vicinal diols and/or glycogen. For this purpose, sections were treated with 1% periodic acid

7
during for 15min. Then, they were washed with running water and dyed during 2 min with

the Schiff reactive (Mc Manus, 1948).

2.4.2 α-amylase-PAS. Sections were subjected to an enzymatic digestion with α-amylase

in a 36º moist chamber during 45 min to identify glycogen. Then, the PAS technique was

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applied (Pearse, 1985).

2.4.3 KOH/PA*S (saponification-selective periodic acid-Schiff’s reagent). This

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technique characterizes GCs with sialic acid residues. The saponification reaction (KOH)

SC
was performed with 0.5% potassium hydroxide in 70% ethanol for 30 min at room

temperature. Before staining with the Schiff’s reagent, sections were subjected to a

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selective oxidation with 0.4 mM periodic acid in 1 M hydrochloric acid at 4ºC (Culling et

al., 1976). N
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2.4.4 PA/Bh/KOH/PAS (periodic acid-reduction with borohydride- saponification-
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periodic acid-Schiff’s reagent). This technique demonstrates the presence of CGs with

sialic acid residues with O-acyl substitutions in 7C, 8C o 9C and O-acyl sugars. Sections
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were subjected to a room temperature oxidation with 1% periodic acid for 2 h. The
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aldehydes generated by the initial oxidation were reduced to primary alcohols with sodium
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borohydride. After saponification (KOH) the PAS technique was applied (Reid et al.,

1973).
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2.4.5 KOH/PA*/Bh/PAS (saponification-selective periodic acid-reduction with

borohydride-periodic acid-Schiff’s reagent). This technique allows the identification of

A

neutral sugars. Sections were treated with 0.5 % potassium hydroxide in 70 % ethanol for

15 min at ambient temperature. Before issuing the PAS technique a selective periodic

oxidation at 4ºC during 1 h followed by a reduction with sodium borohydride was

performed (Volz et al., 1987).

8
 2.4.6 Alcian Blue (AB). AB solutions at different pHs were used to selectively

differentiate color subgroups of acid mucins. Glycoconjugates with carboxylic groups and

O-sulphated esters became evident with a pH 2.8 solution, while sulpho-mucins and highly

sulphated GCs were identified with pH 1.0 and 0.5 solutions, respectively (Lev and Spicer,

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1964).

2.4.7 AB/ PAS (Alcian Blue/ Periodic Acid-Schiff’s Reagent). This combined technique

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allows the identification of acid (AB positive), neutral (PAS-positive) and mixed (AB/PAS

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positive) GCs in the same cut. Sections were exposed to the AB technique followed by the

PAS technique. The AB solution was used at pHs 2.8 and 1.0 to identify carboxylated and

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sulphated GCs, and GCs with O-sulphated esters, respectively (Mowry, 1963).

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2.4.8 Toluidine Blue (TB). The TB solution was used at pHs 5.6 and 4.2 to identify both
A
carboxylated and sulphated GCs, and O-sulphated esters CGs, respectively. In both cases,
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the CGs showing high concentrations of anionic groups stain red-purple (metachromatia);

in the absence of polyanions they stain blue (orthochromasia) (Lison, 1953).

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Results were evaluated by independent observers at a semi-quantitative scale to

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determine the intensity of the reactions (0, negative; 1, light; 2, moderate; 3, strong). This
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classification was established according to previous histochemical studies (Tano de la Hoz

et al., 2014; 2017).

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2.5 Lectin histochemical Study

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A battery of seven biotinylated lectins (Vector Laboratories, Inc. Burlingame, CA, USA)

was used to identify specific sugar residues (Table 1). Paraffin sections were mounted on

slides treated with poly-L-lysine (Sigma Diagnostics, St Louis, MO, USA), deparaffinized

with xylol and incubated in a 0.3% H2O2 solution in methanol for 30 min at room
9
temperature. Then, sections were hydrated, washed with 0.01 M PBS, pH 7.6 and incubated

with a bovine serum albumin in PBS for 20 min. Next, they were independently incubated

with each of the biotinylated lectins for 30 min at room temperature and treated with the

avidine-biotine-peroxidase complex (ABC) during 45 min (Vector Laboratories, Inc). The

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peroxidase complex was activated after 4-10 min incubation with a Tris-HCl 0.05 M, pH

7.6 buffered solution containing 0.02% diaminobenzidine (DAB) (Dako, Carpinteria, CA,

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EE.UU.) and 0.05% H2O2. All lectins were used at a concentration of 30 mg ml-1 in PBS

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dilution, except for PNA that was prepared as 10 mg ml-1.

A semi-quantitative evaluation of the results using the same scale described at 2.4 was

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performed. Two types of controls were used: (1) the lectin solution replaced by PBS and (2)

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lectins pre-incubated for 1 h at room temperature in the presence of the appropriated
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blocking sugars.
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3. Results
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3.1 Morphological and ultrastructural characteristics

The histological structure of the descending colon and rectum of L. maximus showed the
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four typical tunica of the digestive tube. From the luminal surface to the exterior, mucosa,
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submucosa, muscular and serosa tunica were identified. The most conspicuous

characteristic of the descending colon and rectum was the large quantity of folds in the
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wall, formed by a submucosa center, which gives an irregular aspect to the intestinal lumen

of both organs (Fig. 1A, B).

The ultrastructural study allowed the characterization of absorptive and goblet cells from

the intestinal epithelium (Fig. 2). The absorptive cells of both anatomical regions presented

10
a well-defined morphological polarity through the display of microvilli in their apical

surface and junctional complexes in the lateral region of the membrane (Fig. 2A, C). All the

enterocytes presented an electron-lucid cytoplasm and displayed an euchromatic nucleus

and cisterns from the rough endoplasmic reticulum in the basal third. Mitochondria were

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located in the apical cytoplasm between the terminal veil and the nucleus (Fig. 2A-C).

Goblet cells were distributed all along the crypt axis, although they predominated in the

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deep portion of the intestinal gland (Fig. 2D, E). These unicellular glands showed

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microvilli and abundant electron-lucid mucinogen granules concentrated in the apical

cytoplasm.

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3.3 Histochemical study N
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All goblet cells from the descending colon showed carboxylated and sulphated GCs
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(Figs. 3A, B). Even though, neutral secretion cells were identified through the AB pH

2.8/PAS method, mucins of most goblet cells exhibited an acid component (AB positive)
D

(Fig. 3C). Cells of acid and mixed secretions were identified in the middle and inferior
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region of the Lieberkühn crypts, while positive PAS cells were found only in the upper
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third of the intestinal glands (Fig. 3C). The KOH/PA*/Bh/PAS technique also allowed to

corroborate that the pattern of neutral mucins varies along the intestinal crypt axis, being
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the cells of the upper region of the glands those which presented a more intense reaction

(Fig. 3D). The AT method at both pH values revealed the presence of metachromatic goblet
A

cells all along the intestinal gland axis (Fig. 3E, F). Also, scarce sialic acid residues slightly

O-acetylated were identified in the goblet cells of this anatomical region (Fig. 3G, H).

The glycosylation pattern of the rectum was similar to that of the descending colon (Fig.

4A-E); the main difference lay in the fact that the most distal region of the large intestine
11
showed a greater proportion of sialomucins, characterized by being slightly O-acetylated

(Fig. 4F). The glycocalyx of both anatomical regions showed a moderate reaction with the

AB pHs 2.8 and 0.5 techniques. Therefore, it presents carboxylated GCs and sulphomucins

(Figs. 3A-C and 4B-D).

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The histochemical profile of the descending colon and rectum are shown in Tables 2 and

3, respectively. Differences between sexes and capture time were not observed.

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3.4 Lectin histochemical study

The lectin histochemical method evidenced different sugar residues present in the

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glycocalyx, enterocytes and goblet cells from the descending colon and rectum of L.

maximus (Table 4). N

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In both anatomical regions, the glycocalyx presented an intense positive reaction with
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lectins WGA, RCA-I and PNA, while it exhibited no positive staining with UEA-I (Fig.5

A-H). On the contrary, the affinity of Con-A, DBA and SBA varied according to the
D

studied intestinal region. Lectins DBA and SBA showed affinity only for the descending
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colon glycocalyx (Fig. 6A-D), whereas Con-A marked the rectum glycocalyx more
EP

intensely (Fig. 6E, F).

The enterocytes showed a similar glycosylation pattern in both anatomical sectors. The
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most intense marking was detected with lectins WGA and SBA in the supranuclear region

of the cytoplasm (Figs. 5b and 6a).

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Goblet cells of both intestinal regions did not stain with lectins Con-A, DBA and UEA-I.

Therefore, their secreting mucins lack the terminal α-mannose, α-glucose, N-acetyl

galactosamine and L-fucose residues (Figs. 5G, H and 6C-F). Except for WGA, the

remaining lectins demonstrated that the lectin histochemical profile of goblet cells varies
12
according to the intestinal region under study. The main differences encountered between

both anatomical regions were detected in the β-D-N acetylgalactosamine and β-galactose

residues (Figs. 5C, D and 6A, B).

Lectin labelling was completely inhibited when the lectins were omitted from the

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incubation medium or when they were pre-incubated with the appropriate hapten sugar

(Figs. 6G, H). Differences between sexes and capture time were not observed.

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4. Discussion

4.1 Glycosylation pattern of intestinal mucins

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The morphology and ultrastructure of the small and large intestines have been studied in

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several mammalian species (Hosoyamada and Sakai, 2007, Zanuzzi et al., 2008, Hansen et
A
al., 2009, Mantani et al., 2014, Vásquez Cachay et al., 2014). In these studies, it was
M

demonstrated that the epithelium has highly specialized cells, a fact that is related to the

multiple digestive, absorptive, endocrine and immunological functions of the intestinal

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mucosa.
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In L. maximus, as in other vertebrates, the absorptive cells or enterocytes exhibited a

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marked cellular polarity, since they presented uniform microvilli in the apical region and

junctional complexes and interdigitated cytoplasmic processes on their lateral surfaces.

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This shows that, as suggested by Takashima et al., (2013), the membrane specializations

present in this cell type are highly conserved and are associated with absorption and
A

transport processes.

Comparing the ultrastructural characteristics of the epithelial cells of the small and large

intestines of L. maximus, the main differences were observed in the enterocytes. In some

sectors of the small intestine two morphological types of enterocytes were described that

13
differed mainly in the electron-density of their cytoplasm (Tano de la Hoz et al., 2016). In

contrast, in the descending and rectum colon only absorptive cells with electron-lucid

cytoplasm were identified. Although in mammals different types of absorptive cells have

not been identified at the ultrastructural level, in humans, molecular studies have described

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two types of enterocytes in the small intestine that were classified as non-absorptive and

absorptive enterocytes (Gassler et al., 2006). Although future studies are required, the

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results described at the small intestine of L. maximus could offer ultrastructural support to

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the molecular characterization carried out by Gassler et al. (2006). Therefore,

morphological differences found between the enterocytes in the present study could be

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indicative of the existence of cells with different physiological states in the small intestine,

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while in the large intestine of L. maximus this cell type would only fulfill absorptive
A
functions.
M

The intestinal mucins are synthesized and secreted mainly by unicellular epithelial

glands called goblet cells. For this reason, studies carried out in recent years have focused
D

on the knowledge of their morphology, function, distribution and glycosylation patterns

TE

(McDole et al., 2012). As in all mammals, the goblet cells of L. maximus were distributed
EP

throughout the epithelium of the intestinal tract, their number being greater in the large

intestine (Boonzaier et al., 2013; Tano de la Hoz et al., 2014; 2016). This observation using
CC

TEM confirmed the presence of a large accumulation of mucus granules in the apical

cytoplasm, which distends this region of the cell. The presence of abundant mucus granules
A

is an ultrastructural characteristic that is currently used as a criterion to identify cells in a

mature secretory stage (Birchenough et al., 2015). In the large intestine of L. maximus,

mature goblet cells in the lower third of Lieberkühn's glands were characterized by

histochemical and TEM techniques. These observations corroborate that goblet cells of this
14
region of the intestine of L. maximus begin to mature in the area of cellular replication, as is

observed in other vertebrates.

The glycoconjugate analysis demonstrated that the histochemical profile of goblet cells

varies between the descending colon and the rectum of L. maximus, and that it remains

PT
constant in all the studied individuals. These results agree with previous studies describing

gradual variations along the small intestine of L. maximus, and even abrupt changes at the

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level of the colonic groove of the ascending colon were identified (Tano de la Hoz et al.,

SC
2014; 2016; 2017). Altogether, these results demonstrate that each anatomical region of the

intestinal tract of L. maximus shows a particular glycosylation pattern that in turn stays

U
highly preserved between members of the same species. Histochemical and proteomic

N
studies have described similar results for diverse mammal species (Robbe et al., 2004;
A
Boonzaier et al., 2013). Since the histochemical profile of intestinal mucins does not vary
M

among individuals of the same species, some authors have recently proposed that such a

pattern can act as a selection mechanism of the intestinal microbiota (Holmén Larsson et
D

al., 2009; Hansson, 2012). In this way, the glycosylation pattern of the intestinal mucins
TE

would probably be the result of a co-evolutive process between the bacterial flora and the
EP

host in order to maintain an optimal symbiotic relation (Johansson et al., 2011a; 2011b),

being, in turn, a system susceptible to alterations according to age, diet and certain
CC

physiological and pathological states (Beyaz and Liman, 2009; Liquori et al., 2012;

Mastrodonato et al., 2014).

A

As in previous histochemical studies, the AB pH 2.8/PAS technique allowed to evaluate

the existence of goblet cells with different glycosylation patterns (Tano de la Hoz et al.,

2014; 2016; 2017). Thus, in both sectors of the large intestine of L. maximus, cells with

three different histochemical profiles – neutral, acid and mixed– were identified, those with
15
acid secretions being in the largest proportion. These results, together with previous studies,

demonstrate that along the intestinal tract of L. maximus an increasing gradient of

sulphomucins exists, associated to a decreasing gradient of mucins with oxidizable vicinal

diols (Tano de la Hoz et al., 2014; 2016; 2017). Studies on different mammal species have

PT
described a glycosylation pattern similar to that of L. maximus. These researches agree that

there is a predominance of acid mucins, sulphated as well as carboxylated, in the different

RI
large intestine segments (Robbe et al., 2004; Boonzaier et al., 2013). On the other hand, the

SC
existence of goblet cells with different glycosylation patterns demonstrates that there are

functionally different subclasses of goblet cells within a morphologically homogeneous cell

U
population, both at histological and ultrastructural levels. These subpopulations of goblet

N
cells have also been described in the human and rat colonic epithelium by using antibodies
A
against diverse types of intestinal mucins (Podolsky et al., 1986; Gouyer et al., 2011).
M

Since the glycosylation pattern of the intestinal tract acts as a dynamic system able to

adapt to local physiological requirements, including changes in the intestinal microflora

D

(Moran et al., 2011; Pelaseyed et al., 2014), the sulphomucin gradient found along the
TE

intestinal tract of L. maximus would probably be linked to differences in the microbial

EP

charge between the small and large intestine. In this way, these results would also agree

with other studies on rodents, where it was demonstrated that the microflora modulates
CC

specifically the intestinal glycosylation pattern, both at the cellular and subcellular levels

(Freitas et al., 2002; Moran et al., 2011).

A

In mammals, the negative charge of mucins is chiefly determined by sulphate groups

and terminal sialic acid residues (Liquori et al., 2012; Mastrodonato et al., 2014). The high

density of negative charges in intestinal mucins attracts water, thus forming a highly

hydrated gel with viscoelastic properties (Accili et al., 2008). Sialomucins have been
16
identified in the descending colon as well as in the rectum of L. maximus, although in

smaller proportion than sulphomucins. Similar results were described for the mice intestinal

tract by Mastrodonato et al. (2013). Studies on humans instead have reported intestinal

mucins highly sialylated, with little sulphated residues (Robbe et al., 2004). Unlike the

PT
pattern described in humans, our results indicate that the main groups responsible of

determining the acid gradient of mucus along the intestinal tract are those of sulphates.

RI
The lectin histochemical technique has been applied to in situ locate and identify

SC
different terminal and subterminal residues of monosaccharide sugars present in the GCs of

diverse species (Scillitani et al., 2007; Fayed et al., 2010; Mastrodonato et al., 2014). This

U
technique allowed us to characterize the lectin histochemical pattern of the glycocalyx, the

enterocytes, goblet cells and ICC of L. maximus. N

A
In the present study, the goblet cells showed a negative reaction with lectins DBA and
M

UEA-I, regardless of the anatomical region taken into account, which indicates that both

regions lack α-N-acetylgalactosamine and L-fucose terminal residues. Instead, the affinity
D

of the rest of the lectins employed by these cells varied considerably according to the
TE

intestinal segment. In agreement with our results, Galotta et al. (2009) demonstrated that
EP

the lectinhistochemical pattern of goblet cells showed a large variation depending on the

intestinal region studied.

CC

In mammals, the presence of N-acetylglucosamine (GlcNAc) is particularly associated

with the acidification degree of mucins, for the sulphate groups are generally linked to this
A

monosaccharide (Liquori et al., 2012). Lectin WGA revealed terminal residues in goblet

cells in the entire intestinal tract of L. maximus, having the ascending colon and the rectum

the greatest marking intensity (Tano de la Hoz et al., 2014; 1016; 2017). In this manner, in

17
the present study a correspondence between the acid gradient of secreting mucins and the

distribution of N-acetylglucosamine terminal residues was found.

The secreting function of the enterocytes consists of the synthesis of glycoprotein

enzymes that will be inserted in the apical cell membrane. Biosynthesis of glycan chains

PT
mainly occur in compartments of the rough endoplasmic reticulum - Golgi in reactions

implying the sequential activity of glycosidases and glycosyltransferases (Moran et al.,

RI
2011). In the present study, the enterocytes of most of the intestinal sectors evidenced a

SC
positive supranuclear marking with some of the used lectins. As demonstrated in other

lectin histochemical studies, it is probable that this marking pattern be indicative of the

U
glycosylation process undergone by CGs within the endomembrane system (Gabrielli and

N
Tomassoni, 2018). As a morphological correlate, in these absorptive cells, Golgi cisternae
A
in the supranuclear region and a well-developed rough endoplasmic reticulum were
M

observed.
D

Declarations of interest
TE

None.
EP

Acknowledgments
CC

This research was supported by a Grant from Universidad Nacional de Mar del Plata

(EXA 765/16), Buenos Aires, Argentina. We would like to thank to the staff of Estación de
A

Cría de Animales Silvestres (ECAS), Ministerio de Agroindustria de la Provincia de

Buenos Aires.

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24
 PT
RI
SC
U
Fig. 1. Histological characterization of the descending colon and rectum. (A) Low
N
magnification image of the descending colon in which the characteristic folding of its wall
A
is visible, H-E. (B) Detail of a fold of the rectal wall, Masson's trichrome. Asterisk, lumen;
M

LP, lamina propria; mm, muscularis mucosae; Muc, tunica mucosa; SubM, tunica

submucosa; TM, tunica muscularis. Scale bar: 300 µm (A); 100 µm (B).
D
TE
EP
CC
A

25
 PT
RI
SC
U
N
A
M
D
TE

Fig. 2. Transmission electron micrographs of enterocytes (A-C) and goblet cells (D, E). (A)

Ultrastructural characteristics of enterocytes. (B) Inset magnification on the A showing the

EP

enterocyte’s perinuclear cytoplasm. (C) High magnification image of the intercellular

junctions between two neighboring cells. (D, E) Goblet cells at the bottom of a crypt.
CC

Arrowhead, desmosome; G, secretory granules; jc, junctional complex; M, microvilli; mi,

A

mitochondrion; N, nucleus; rER, rough endoplasmic reticulum. Scale bar: 2 µm (A, B, D,

E); 1 µm (C).

26
 PT
RI
SC
U
N
A
M
D
TE
EP
CC

Fig. 3. Glycosylation pattern of the epithelium of the descending colon. (A) AB pH 2.8. (B,
A

b) AB pH 1,0. (C) AB pH 2.8/PAS. (D) KOH/PA*S. (E) AT pH 5.6. (F) AT pH 4.2. (G)

PA/Bh/KOH/PAS. (H) KOH/PA*/Bh/PAS. Arrow, glycocalyx; GC, goblet cell; GCa,

goblet cell PAS positive; GCb, goblet cell AB/PAS positive; GCc, goblet cell AB positive;

mm, muscularis mucosae. Scale bar: 20 µm (A-H); 15 µm (b).

27
 PT
RI
SC
U
N
A
M
D
TE

Fig. 4. Glycosylation pattern of the rectal epithelium. (A) PAS. (B) AB pH 2.8/PAS. (C)

AB pH 2.8. (D) AB pH 1.0/PAS. (E) AT pH 5.6. (F) KOH/PA*S. Arrow, glycocalyx; GC,
EP

goblet cells; GCa, goblet cell PAS positive; GCb, goblet cell AB/PAS positive; GCc, goblet
CC

cell AB positive. Scale bar: 20 μm.

A

28
 PT
RI
SC
Fig. 5. Lectin binding profile of the descending colon (A, C, E, G) and rectum (B, b, D, F,

H). (A, B, b) WGA. (C, D) RCA-I. (E, F) PNA. (G, H) UEA-I. Arrow, glycocalyx;

U
arrowhead, supranuclear granules; GC, goblet cell. Scale bar: 20 µm (b, C- H); 40 µm (A,

B). N
A
M
D
TE
EP
CC
A

29
 PT
RI
Fig. 6. Lectin binding profile of the descending colon (A, C, E, G) and rectum (B, D, F, H).

SC
(A, a, B) SBA. (C, D) DBA. (E, F) Con-A. (G, H) Controls performed by substitution of

U
the lectin solution with PBS (G) and preincubation of the sections with the corresponding

N
hapten sugar (H). Arrow, glycocalyx; arrowhead, supranuclear granules; GC, goblet cell.
A
Scale bar: 20 µm (a, B, D-H); 40 µm (A, C).
M
D
TE
EP
CC
A

30
Table 1. Lectins used and their carbohydrate specificities

Lectin Acronym Specificitya Haptene

Group I Glc/Man
Canavalia ensiformis Con-A α-D-Man; α-D-Glc α-D-Methyl-Man
agglutinin

PT
Group II GlcNAc
Triticum vulgaris WGA β-D-GlcNAc; NeuNAc NeuNAc

RI
(wheatgerm)
agglutinin

SC
Group III GalNAc/Gal
Dolichos biflorus DBA α-D-GalNAc D-GalNAc

U
agglutinin
Glycine maximus
agglutinin
SBA N
α-D-GalNAc; β-D-GalNAc D-GalNAc
A
Ricinus communis RCA-I β-Gal Lactose
M

Agglutinin-I
Arachis hypogaea PNA β-D-Gal (β1-3) > D-GalNAc Lactose
D

agglutinin
TE

Group IV L-Fuc
Ulex europaeus UEA-I α-L-Fuc L-Fuc
Agglutinin-I
EP

a
Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-
CC

acetylglucosamine; L-Fuc, L-fucose; Man, mannose; α-D-Methyl-Man, α-D-Methyl-

mannose; NeuNAc, acetyl-neuraminic acid (sialic acid).
A

31
Table 2. Histochemical analysis of the descending colon of Lagostomus maximus

Procedures Glycocalyx Enterocyte Goblet cells

Upper crypt Medial crypt Lower crypt

PAS 0 0 2 1 1
α-amylase/PAS 0 0 1 1 1

PT
KOH/PA*S 0 0 2 2 2
PA/Bh/KOH/PAS 0 0 1 1 1
KOH/PA*/Bh/PAS 1 0 3 2 2

RI
AB pH 2.8 1 0 3 3 3
AB pH 1.0 3 0 3 3 3

SC
AB pH 0.5 0 0 1 1 1
AB pH 2.8/PAS 1T 0 3T-3P-2Ma 3T-3Pa 3T-3Pa
AB pH 1.0/PAS 1T 0 3T-3P-2Ma 3T-3Pa 3T-3Pa

U
TB pH 5.6 2or 2or 3m 3m 3m
TB pH 4.2 0 0 3m 3m 3m
N
m, metachromasia; M, magenta; or, ortochromasia; P, purple.
A
Staining intensity: 0, negative; 1, slightly positive; 2, moderate; 3, strong.
a
Goblet cells with two different histochemical profiles were differentiated.
M
D
TE
EP
CC
A

32
Table 3. Histochemical analysis of the rectum of Lagostomus maximus

Procedures Glycocalyx Enterocyte Goblet cells

Upper crypt Medial crypt Lower crypt

PAS 0 0 2 1 1
α-amylase/PAS 0 0 1 1 1

PT
KOH/PA*S 0 0 2 2 2
PA/Bh/KOH/PAS 0 0 1 1 1
KOH/PA*/Bh/PAS 1 0 3 2 2

RI
AB pH 2.8 3 0 3 3 3
AB pH 1.0 3 0 3 3 3

SC
AB pH 0.5 0 0 1 1 1
AB pH 2.8/PAS 1T 0 3T-3P-2Ma 3T-3P-1Ma 3T-3P-1Ma
AB pH 1.0/PAS 1T 0 3T-3P-2Ma 3T-3P-1Ma 3T-3P-1Ma

U
TB pH 5.6 2or 2or 3m 3m 3m
TB pH 4.2 0 0 3m 3m 3m
N
m, metachromasia; M, magenta; or, ortochromasia; P, purple.
A
Staining intensity: 0, negative; 1, slightly positive; 2, moderate; 3, strong.
a
Goblet cells with different histochemical profiles were differentiated.
M
D
TE
EP
CC
A

33
 Table 4. Lectinhistochemical analysis of the descending colon and
rectum of Lagostomus maximus.

Lectin Descending colon Rectum

Con-A Glycocalyx 1 2

Enterocyte 1 1

PT
Goblet cells 0 0

WGA Glycocalyx 3 3

RI
Enterocyte 3a 3a

SC
Goblet cells 3 3

DBA Glycocalyx 2 0

U
Enterocyte 0 0

Goblet cells N
0 0
A
SBA Glycocalyx 3 0
M

Enterocyte 3a 3a

Goblet cells 0-3b 0-2b

D

RCA-I Glycocalyx 3 3
TE

Enterocyte 0 0

Goblet cells 2 1
EP

PNA Glycocalyx 3 3

Enterocyte 1 0
CC

Goblet cells 2 1
A

UEA-I Glycocalyx 0 0

Enterocyte 0 0

Goblet cells 0 0

Staining intensity: 0, negative; 1, slightly positive; 2, moderate; 3, strong.

34
a
Only the supranuclear region of the enterocytes was labeled.
b
Goblet cells with two different histochemical profiles were differentiated.

PT
RI
SC
U
N
A
M
D
TE
EP
CC
A

35
Another random document with
no related content on Scribd:
Humber, to be carried eastward through Lincolnshire, into the East
sea.
I presently suspected, this was owing to the artificial cut of the
Romans, called Fossdike, part of the Carsdike; which Fossdike is
drawn from Torksey at the Trent, to Lincoln: there it meets the river
Witham coming from the south, and proceeds eastward toward
Boston.
Ever since I was capable of observation, I often took notice, that
the whole flat, or fenny country of Lincolnshire, has a gentle declivity,
or natural descent eastward. This is owing not only to the sea lying
that way, but is the case of all levels in the whole globe: the cause
must be asserted to be the earth’s rotation upon its axis; which
observation I printed, long since, in my Itinerarium Curiosum.
It is a principle in nature, that, when a globe is turned on its axis,
the matter on the surface flies the contrary way to its motion. The
philosophers call this improperly a conatus recedendi ab axe motus:
it is not owing to an endeavour of matter to fly the contrary way, but
to the innate inactivity of matter that resists the motion; does not
readily follow it.
But it is evident from hence, that the earth, receiving its motion
before the surface was perfectly consolidated, the moistish matter
would be left westward, as far as it could be, and produce an
extended and gentle declivity on the east; and at the same time, by
stiffening, would render the west side of all hills steep.
This is a fact throughout the whole globe. Hence it is, that all
plains and levels have naturally their descent towards the east; and
hence it is, that the river of Witham, from Grantham side, running
northward to Lincoln, readily takes its course thence eastward, to
meet the ocean over the fenny level.
The Romans, when they made the artificial canal, the Carsdike,
from Peterborough along the edge of the Lincolnshire fens,
introduced it into the river Witham, three miles below Lincoln. The
purpose of this artificial cut was, to convey corn in boats, from the
southern parts of England, to the northern prætentura’s in Scotland
for maintenance of the forces kept there: therefore the canal,
entering the Witham, passed through Lincoln, and then was
continued by another artificial cut, called the Fossdike, from Lincoln
to Torksey, where it enters the Trent, in order to go down the stream
to the Humber: from thence the fleet of corn-boats passed up the
river Ouse to York, by force of the tide; for so high will the tide carry
them; which was the reason of building the city there.
After this Fossdike, between the Trent and Witham rivers, was
made by the Romans, it is easy to imagine, that the extensive river
of Trent, which runs altogether northwards, would very readily, upon
great floods, discharge part thereof into the Fossdike; for there is a
descent that way, as being to the east: and this might be the
occasion of the geography in our map, mistaking the Fossdike, and
the continuation of the Witham, for that of the Trent.
The river Witham, from Lincoln, goes south-east into the sea, by
Boston; and it seems to me, that in very early times it might (at least
in great floods) have another channel running over the East fen (as
called) along that natural declivity, full east, into the sea, as in the
map of Richard of Cirencester.
This channel might pass out of the present river of Witham a little
below Coningsby, where the river Bane falls into it, at Dockdike and
Youldale, by the water of Hobridge, north of Hundle-house; so
running below Middleholm to Blacksike, it took the present division
between the two wapentakes, all along the south sides of the deeps
of the East fen; and so by Blackgote to Wainfleet, the Vainona of the
Romans.
My friend, John Warburton, Esq; Somerset herald, has some
manuscripts of our Lincolnshire antiquary, some years ago, Mr. De la
Pryme, who was perfectly acquainted with that part of Lincolnshire,
and therein discovers some suspicions of the Trent running toward
Lincoln in antient days; but I think, all we can certainly conclude from
our map is the extreme antiquity of it: as the Carsdike must have
been projected and done by Agricola, on his conquest of Scotland,
we may reasonably judge this to be in the main his map, i.e. copied
from his, though with some additions by our author.
 This consideration, duly attended to, shows the antiquity of the
Fossdike, and Carsdike, and of our map.
We are told in the History of Carausius, that he repaired the
prætentura made in Scotland by Agricola, and added seven forts to
it: a wise and politic prince knew the necessity of it; and
consequently infer we, that he as surely repaired the Carsdike
navigation, to supply the soldiers with corn, in that northern situation:
and I have several reasons to induce me to conclude, he not only did
so, but carried it further southward than before, viz. from
Peterborough quite to Cambridge; some of which reasons I shall
recite in the history of that hero. At present I shall only hint, that his
name has ever been affixed to this famous canal, which has never
been regarded by writers. It is of utmost importance in the
knowledge of Roman antiquity; and it is an affair of such public
emolument, as not to be unworthy of the notice of the legislature;
where an inland water-carriage is made, for 200 miles in length, from
Cambridge to Boroughbridge.
The Roman provinces, as we find them in our map, are these.
Maxima Cæsariensis, or Brittania superior, chiefly the country of the
Brigantes, conquered by Cerealis, and so named by him, in the
beginning of Vespasian’s reign.
Valentia, all that country comprehended between the two
Prætentura’s.
Brittania prima, or inferior, that part of the island south of the
Thames.
Brittania secunda, being Wales.
Flavia Cæsariensis, that part between the Humber and the
Thames; denominated from the family-name of Vespasian.
Vespasiana, that part of Scotland between the Varar Æstuary, or
highland boundary, and the northern Prætentura.
Lastly, Caledonia properly, or the Highlands, which the Romans
never conquered; and that part called Vespasiana, after Agricola
returned, was neglected by Domitian, and recovered by the Scots; at
least, to the first Prætentura: and it is from Richard of Cirencester
alone, that we have an Itinerary of it from the Vararis Æstuary, on
which is the last Roman station, called Alata castra, now Inverness.
I shall next recite all the places, rivers, mountains, &c. specified in
our map, the provinces they are in, and that in alphabetical order;
together with the modern names of each, according to the best of my
knowledge; whereby the value and excellence of our manuscript will
more easily appear; seeing so many of them we were hitherto
unacquainted withall, which I shall mark particularly thus *, as also
those wherein we are able to correct former writers.
Places mentioned in the Map.
* Abona fluvius Caledoniæ, Frith of Dournoch.
Abona fl. Brittaniæ Primæ Provinciæ, Avon by Bath.
Abus fl. the Humber.
* Albanii, Broad albin.
Alauna, Sterling.
* Alpes, Valentiæ Provinciæ, hills of Lothlers.
Alauna fl. Aylemouth, Northumberland, Awne.
* Alauna fl. Maximæ, Lune r. of Lancaster.
Alauna, Flaviæ, Aulcester upon Arrow r. Warwickshire.
Alauna fl. by Blandford, Dorsetsh.
Antona fl. Avon, or Nen of Northampton.
Antivestæum Promontorium, Penros, Cornwall.
Anderida, Newhaven, Sussex.
* Aræ finium Imperii Romani, Chanary.
Artavia, Tintagel, C. Cornwall.
Ariconium Secundæ, Kenchester, Herefordshire.
* Attacotti, Vespasianæ Provinciæ, Lochabar.
Atrebates, Berkshire people.
* Aquæ, Buchan.

Banatia, Vespasianæ, by Fort-William, Lochabar.

Banchorium, Banchor.
 Berigonium, Valentiæ, Dunstafag, in Lorn.
* Berigonius finus, by Cantyre.
Belisama fl. Maximæ Cæasariensis, Rible r. Lancashire.
Benonæ, Highcross, Northamptonshire.
* Bibrax, Madanhead, Bray, Berkshire.
Bodotria æstuarium, Frith of Forth.
Boduni, Oxfordshire and Gloucestershire.
Bolerium prom. Primæ, St. Ives, Cornwall.
Bremenium, Rochester, Northumberland.
Brigantes, Yorkshire men.
* Brigantum extrema, Flamborowhead, Yorkshire.
Brangonium, Flaviæ Provinciæ, Worcester.

* Caledoniæ extrema, Caledoniæ, Dungsby head.

Caledonii, Inverness county.
Caleba Attrebatum, Wallingford, Berkshire.
Cambodunum, Latio jure donata, Alkmonbury.
* Camboritum colonia, Chesterford, Cambridgeshire.
Camulodunum colonia, Colchester, legio gemina martia XIV.
* Cambola fl. Padstow haven, Cornwall; Camelford.
* Cantæ, Kent.
* Cantiopolis, Primæ, Canterbury; stipendiaria.
* Canganus sinus, by Harley, Merionidshire.
Cantæ, Cromarty.
Candida casa, s. Lucopibia, Whithern.
Carronacæ, Strathnavern, Carnovacæ.
* Carnabii, Sutherland.
Carbanticum, Kirkcubright, Treefcastle on Dee r.
* Carnabii, Flaviæ, Cheshire and Staffordshire.
Cassii, Middlesex.
Cassiterides ins. Scilly islands.
Cataracton, Maximæ, Catteric, Yorkshire; Latio jure donata.
* Cattini, Cathness.
* Cauna ins. Shepey isle.
Celnius fl. Davern r.
Cenia, Tregeny, Falmouth.
Cenius fl. Tregeny, Cornwall; Falmouth haven.
* Cenomani, Huntingdonshire, Cambridge, Suffolk.
Cerones, Inverness county.
* Cimbri, Primæ, Somersetshire.
Clausentum, Southampton.
Clota insula, Vespasianæ, Arran isle.
* Clita fl. Secundæ, Clvyd r. St. Asaph.
Clotta æstuarium, Valentiæ, Cluyd fryth.
Cluda fl. Cluyd r.
* Coccium, Burton n. of Lancaster; Latio jure donata.
Colanica, Valentiæ, Peblis.
Conovius fl. Conovy r. Aberconway.
Coria, Carstownlaw in Lothian.
Corinium Dobunorum, Cirencester.
* Coritani, Leicestershire, Lincolnshire.
* Corium, Corsford in Cluydsdale.
* Creones, Ross.

* Damnii, Valentiæ, Lorn.

Damnii, Vespasianæ, Argyleshire.
Damnonii, Primæ, Somersetshire.
* Dena fl. Cree r. by Withern.
Derventio fl. Maximæ, in Cumberland.
Derbentio, Little Chester by Derby.
Deva fl. Dee r. by Kirkcubright.
Deva colon, leg. creticæ XX. V. V. Flaviæ, Dee r. W. Chester.
Deva fl. Dee r. of Aberdeen.
* Dimeti, Secundæ, Cardiganshire.
* Durius fl. Dart r. Devonshire.
* Durinum, Dorchester, Dorsetshire.
Durobris, Rochester.
Dubris, Dover.
* Durnomagus, Caster by Peterborough; Latio jure donatus.

Eboracum, municipium, York, formerly a colony of leg. VI.

Ebuda ins. Caledoniæ, Hebrid islands.
* Epidia ins. superior, Vespasianæ, Northvist. ins. inferior,
Southvist.
* Epidii, Cantyre.
* Epiacum, Maximæ, Chester in the Street.
Etocetum, Flaviæ, Wall by Litchfield.

* Forum Dianæ, Market Street, by Dunstable.

* Fretum Menevicum, Secundæ, Cardigan bay.

Gadeni, Valentiæ, in Northumberland.

* Galgacum, Maximæ, Lanchester, Durham county.
Garion fl. Garienus, Yare, velox.
Glevum Flaviæ, Glocest. colonia leg. Claud. VII.
Gobanium, Secundæ, Abergavenny.
Grampius m. Vespasianæ, Grantsbein.

* Halengum, Hailston, Cornwall.

* Hedui, Somersetshire.
* Helenum prom. Berry point, Devonshire.
* Hereclea ins. Primæ, Lundy isle.
Herculis prom. Hertford point, Devonshire.
* Heriri m. m. Secundæ, Wales.
Horestii, Vespasianæ, Fife.
 Icenii, Flaviæ, Rutlandshire.
Idumanus fl. by Chelmsford.
Ila fl. Caledoniæ, Ale r.
Isca fl. Primæ, Ex by Exeter.
Isca Dumnoniorum, Exeter.
Isca colon. Silurum, leg. Secundæ, Aug. Caerleon.
Isca fl. Uske r. Monmouthshire.
Isurium Brigantium, Maximæ, Aldwark by Burrow-bridge.
Ituna fl. Vespasianæ, Ythan r.
* Ituna æst. Valentiæ, Eden.

* Κριου μέτωπον, prom. Primæ, Ramhead.

Lelanonius sinus, Vespasianæ, Loch luven.

Lemanus, Primæ, Limne, Portus.
Lemana fl. Lime water.
* Lincalidor lacus, Loch lomund.
* Lindum, Dunblain.
Lindum colon. Lincoln.
* Logi, Sutherland.
Londinium Aug. Flaviæ, London; colonia.
* Longus fl. Loch loch.
* Loxa fl. Caledon. Frith of Cromartie.
* Lucopibia, s. candida casa, Valentiæ, Whitehern.
Lugubalia, Maximæ, Carlisle.
* Luanticum, Secundæ, Cardigan.

Magna, old Radnor.

Maleos ins. Mull isle.
* Mare Orcadum, Pentland fryth.
* Mare Thule, Caledon, the North-British sea.
Mediolanum, Secundæ, Myvod, Montgomeryshire.
* Menapia, St. David’s South Wales.
* Menapia ins. Ramsey isle. Mertæ, Murray.
* Merseja fl. Mersey r. Cheshire.
Metaris æst. Flaviæ, Boston deeps, Washes, Lincolnshire.
Mona ins. Anglesey in North Wales.
* Monada ins. Isle of Man.
* Morini, Somerset and Dorsetshire.
Moricambe fl. Maximæ, Decker r. Lancashire.
* Muridunum, Primæ, Columb, Cornwall.
Muridunum, Caermarthen, South Wales.

Nabius fl. Caledon Navern.

Nidus fl. Nith. r. Nithisdale.
* Nidus fl. Secundæ, Neath r. Glamorg.
Novantæ, Valentiæ, West Galway.
* Noviomagus, Primæ, Croydon.

* Oceanus Deucalidon, Western British sea.

* Ocetis ins. Caledon, Strom. isle.
Ocrinum m. Primæ, Penryn, Cornwall.
Octurupium prom. Secundæ, Bishop and Clerks, Pembrokeshire.
* Olicana, Maximæ, Wetherby on Wherse.
Orcas prom. Caledon. Farro head.
* Orrea, Vespasianæ, Perth, St. Johnston.
* Otys fl. Loch Soil, Lochaber.
Oxellum prom. Spurn head, Yorkshire.

Parisii, Holderness, Yorkshire.

* Penninæ m. m. Maximæ, the Peaks.
* Penoxullum prom. Terbaetness, in Ross.
* Petuarium, Brough on the Humber.
Pomona ins. Caledon. Mainland isle Orkneys.
* Portus fœlix, Bridlington bay.
* Pteroton, alata castra, Vespas. Inverness.

Ragæ, Flaviæ, Ratæ Coritanorum, Leicester.

* Regnum, Chichester.

Sabrina æst. Primæ, Severn.

* Salinæ, Flaviæ, Droitwich, Worcestershire.
* Salinæ, town of Saltwarp, river Saltwarp, Droitwich; a branch of
the Severn.
Segontiaci, about Silchester, Hampshire.
Segontium, Secundæ, Caernarvon.
Selgovæ, Valentiæ, Annandale, Solway frith.
Silures, Herefordshire.
* Silva Caledon. Caledoniæ, Stetadel forest, Sutherland.
* Silva Caledoniæ, Rockingham forest.
* Sistuntii, Maximæ, Lancashire.
Sorbiodunum, Old Sarum.
* Strabo fl. Ouder gill r. Ross.
Stuccia. fl. Rhydel r. by Aberystwth, S. Wales.
* Sturius fl. Stour, r. by Sudbury, Essex.

Taixalorum, prom. Buchan ness.

Tamara, by Tavistoke upon Tamar r.
Tamarus fl. Tamar r. Devonshire.
* Tamea, Brumchest by Blair.
* Tavus æst. Tay frith.
Tavus fl. Tay r. by Perth.
* Tebius fl. Tewy r. by Carmarthen.
* Termolum, Primæ, South Molton, Devonshire.
* Texalum, Castle in Mearns.
Thamesis fl. Thames r.
 Thanatos ins. Thanet isle.
* Theodosia Vespasianæ, Dunbriton.
* Thermæ colon. Bath; Aquæ Solis.
Thule ins. Caledon. Iceland.
Tina fl. by Montrose.
* Tisa fl. Maxim. Tees r. Yorkshire.
* Tobius fl. Secund. now Chymny, by Cardiff.
Trinobantes, Middlesex.
Trisanton fl. Newhaven, Sussex.
* Trivona fl. Flav. Trent r.
Tuæssis, upon Spay r.
* Tuerbius fl. Tyvy r. by Cardigan.

Vacomagi, Vespasianæ, Athol.

* Vaga fl. Secundæ, the Wye r. Herefordshire.
Vallum Severi, the Wall of Severus.
* Vanduaria, Krawford in Cluydsdale.
Varar æstuar. Frith of Murray.
Vecta ins. Wight island.
* Vecturiones, Angus people.
Vedra fl. Weremouth.
Venta Icenorum, Caster by Norwich.
Venta Belgarum, Winchester.
Venta Silurum, Caerwent, Monmouthshire.
* Venta, Wimborn minster, Dorsetshire.
Verolanium, Verlamcester, St. Alban’s; municipium.
* Vervedrum pr. Caledon. Ness head.
Victoria, Airdoch.
* Vidogaræ fl. Valentiæ, Ayr. r. in Kyle.
Vindonum, Silchester, Berkshire.
Vindelis prom. Portland isle, Dorsetshire.
* Vinovium, Piers bridge, Ovynford.
 Virubrium prom. the Ord head, Scotland.
Volsas sinus, Loch breyn in Ross.
Voluba, Grampound, Cornwall.
* Voluntii, Maximæ, Amunder ness hundred, Lancashire.

Uriconium, Flaviæ, Wroxeter, Shropshire.

* Uxella, Barton on the Foss road, Somersetshire.
* Uxella fl. Primæ, by Glastonbury, Somersetshire.
* Uxella m. hills of Lothlers, Cluydsdale.
Uxellum, Dumfrys in Nithsdale.
Uxellum, rightly placed by Baxter, the r. Nyth, Nithisdale, or
Dumfries.
Thus I have recounted the names of places contained in this
excellent map, to the number of 250; whereof 100, marked in this
catalogue thus *, are wholly new, or ill-placed by former writers. The
reader versed in these kind of inquiries, will find no small number of
them; to his judgement I leave them: as to me, the finding fault with
others endeavours is very disagreeable. This I may say; it sets us
right in abundance, wherein before we had no guide but conjecture,
from similitude of names: as, for instance, Uxella, placed in some
great authors at Lestwthiel, Cornwall, is in Somersetshire, viz. at
Barton, where the Roman road called Foss crosses the river, a little
north of Ilchester. Many more might be specified, where only a map
can properly direct us.
I must take notice of another use in our map. In the province of
Brittania Prima are two Venta’s; but till now we could not ascertain
them both: the map shows us, one is Wimborn minster, the other
Winchester: the former is on the river Alauna, seen plainly in
Blandford, being the ford over the Alauna; Llaunford, in the Belgic
pronunciation: called now Allen river. Our author calls Canterbury,
Cantiopolis, though before we knew no other name it had than
Durovernum: but the modern name of Canterbury seems derived
from the former; and the termination favours our author’s
observation, in another part of his history, of remains of Greek
traders preserved in some places; of which several more instances
may be given.
I extend my inquiries here, on Richard of Cirencester’s map, no
further than our island of Britain; leaving that of Ireland to those that
have proper opportunities.
Nor shall I pretend to assign places in Scotland, any further than
the map directs me; but leave them too to those that have proper
opportunities of inquiry, in that kingdom.
 III.

L ET us now proceed to his Itinerary; a truly invaluable monument!

From these two we may hope to obtain a complete knowledge of
Roman Britain.

CAPUT VII.
Our author calls these, Iters of his Diaphragmata, from their
similitude to the animal midriff, passing through the body from side to
side.
Rhutupis colonia, Sandwich, Richborough and Stonar castle, Kent,
is the first city, says our author, in the island of Britain, towards Gaul;
situate among the Cantii, opposite to Gessoriagum, the port of
Bononia, Boloign. Hence is the most commodious passage of ccccl.
stadia, or, as others will have it, xlvi. miles.
From that city Rhutupium, says he, is drawn the Roman way called
Guithlin-street, quite to Segontium, Caernarvon, through the space of
cccxxiv. miles, or thereabouts. Thus,
To Cantiopolis, which is also called Durobernum, stipendiaria,
Canterbury, Kent, x. miles.
Durosevum xii. Sittingburn, Kent.
XXV.
Duroprovis, stipendiaria, Rochester, Kent.
Thence, at xxvii. miles, it passes the Thames, and enters the
province Flavia, and the city of Londinium Augusta, London. Thence
IX.
To Sulloniagis, Suellaniacis, Edgeware, Middlesex.
XII.
 Verolamium, municipium, Verlamcester, or St. Alban’s. Of this
place were Amphibalus and Albanus, martyrs.
XII.
Forum Dianæ, Market street, near Dunstable, Hertfordshire.
XII.
Magiovinium, Dunstable, Bedfordshire.
XII.
Lactorodum, Stoney Stratford, Bucks.
XII.
Isannavaria, Isantavaria, Towcester, Northamptonshire.
XII.
Tripontium, Dowbridge, Stanford, Northamptonshire.
IX.
Benonis, Highcross, Cleycester, between Warwickshire and
Leicestershire. Here the road is divided: the one branch, the Foss,
goes to Lincoln; the other to Viriconium, Wroxeter, from Tripontium.
XII.
To Manduessedum, Mancester, near Atherston, Warwickshire.
XIII.
Etocetum Wall, by Litchfield, Chesterfield wall, Staffordshire.
XII.
Pennocrucium, by Penkridge, Staffordshire.
XII.
Uxoconium, Okenyate, Shropshire.
XI.
Virioconium, Wroxcester, Salop.
XXVI.
Banchorium, Bonium, Banchor, Flintshire.
X.
 Deva colonia, leg. vices. victrix Cretica, Westchester; the border of
Flavia and Secunda provinces.
XXX.
Varis, Bodvary by Denbigh on r. Clwyd.
XX.
Conovium, Aberconway, Carnarvonshire.
XXIV.
Seguntium, stipendiaria, Caernarvon.
Were I to recite all I have written upon this work, by way of
comment, it would amount to a large volume; yet some few remarks I
must make.
What all others call Durolenum our author names Durosevum,
which I affix to Sittingburn, favouring this reading: the distance
conformable.
Sulloniacis, or rather Suellaniacis, has its name from Suellan, or
Cassibelin, who fought Cæsar. I place it at Edgware, which has its
name from the agger, or high raised Roman way, Watling-street.
Here was Cassibelin’s usual residence: his oppidum, or military town,
which Cæsar stormed, was at Watford.
Forum Dianæ, a new name, was crouded into the roll of the
original Itinerary, where the intermediate distance, xii. miles, between
St. Alban’s and Dunstable, remained unaltered: therefore the
transcriber repeated the same distance erroneously.
I doubt not, the place is what we now call Market-street, a little on
this side Dunstable, upon the great road Watling-street. Here was a
fane, and forum, or portico, sacred to Diana; where a panegyre, or
fair, as we call it, was annually celebrated, to the honour of the
goddess, by the lovers of hunting, on the great festival sacred to her,
when stags were sacrificed: this was upon August 13, the hunters’
day, in the Roman kalendar.
I have no need to be ashamed in acknowledging an error incurred
in my juvenile travels, when we knew nothing of this work of our
author’s; for now I apprehend Durocobrivis is another name of a town
near this place: the modern name of Redburn proves it, which means
the same as Durocobrivis, the passage over the Redwater brook.
Rotten row, Rowend, Flamsted by Forum Dianæ, names importing
high antiquity: Rotten row, just by Bremenium, Ruchester; again at
Dorchester, Oxfordshire: they relate to panegyres, or fairs.
Manduessedum, Mancester, on each side the Watling-street, was
walled about.
The vestigia of Benonis are at Claybrook.
Thus we have the whole length of the Watling-street, from Dover to
Caernarvon.

ITER II.
A Segontio, Caernarvon, Virioconium, Wroxcester, usque lxxiii.
miles, thus.
Segontium, stipendiaria, Caernarvon, Carnarvonshire.
XXV.
Herirus mons, Raranvaur hill by Bala, Merionethshire, by
Pimblemere.
XXV.
Mediolanum, Myvod, on Merway r. Montgomeryshire.
XII.
Rutunium, Rowton castle; Stanford, Watlesborough, west of
Shrewsbury.
XI.
Virioconium, Wroxcester on the Severn, below Shrewsbury, under
Wrekin hill.
Caernarvon stands on the river Seint, Seient, Segont, said to have
been built by Constantine the Great. Nennius gives it the name Kaer
Kustenidh, for that reason: he probably made the Via Heleniana, in
honour of his mother, called Sarn Helen.
 Herirus mons has its name from the eagles inhabiting the place,
Celtic.

ITER III.
From Londinium, London, to Lindum colonia. Lincoln, thus,
Londinium Aug. London.
XII.
Durositum, Romford, Essex.
XVI.
Cæsaromagus, Chelmsford, Essex.
XV.
Canonium, Kelvedon, Essex.
IX.
Camulodunum colonia, leg. gem. Mart. Victrix, Colchester, Essex.
VI.
Ad Sturium amnem, ad Ansam, Stretford street, Suffolk.
XV.
Combretonium, Bretenham, Stow, Combe, Suffolk.
XXII.
Sitomagus, Thetford, Norfolk.
XXIII.
Venta Cenomanorum, stipendiaria, Caster by Norwich, Norfolk.
XXVII.
Icianis, Ixworth, Suffolk.
XX.
Camboritum, colonia, Chesterford, Cambridgeshire.
XX.
Durosiponte, Godmanchester, Huntingdonshire.
 XX.
Durnomagus, Latio jure donatus, Dormancester, Caster by
Peterborough, Northamptonshire.
XX.
Causennis, Corisennis, Stanfield by Bourn, Lincolnshire.
XX.
Lindum colonia, Lincoln.
Iter VI. of Antoninus, a Londinio Lindum, goes quite a different way
from this; the one to the right, the other to the left of the straitest way,
the Hermen-street. Instead of our Durnomagus on the northern, he
mentions Durobrivis, Chesterton, on the southern bank of the river
Nen, a walled city: a bridge over the river, built since the time of our
Itinerary. And also
From Camboritum to Durosiponte, in this Iter of ours, and Vth of
Antoninus, I collect, the Roman city of Cambridge, Granta, was not
then in being.
I suppose, it was founded by Carausius, when he carried the
Carsdike from Peterborough to Cambridge, and made the road over
Gogmagog hill from Durosiponte, Godmanchester, to Camulodunum
colonia, Colchester; for all these Itineraries were made before
Carausius’s time.

ITER IV.
From Lindum, Lincoln, to the Vallum, the Roman wall, thus.
Lindum colonia, Lincoln.
XIV.
Argolicum, Littleborough on Trent, Nottinghamshire.
XX.
Danum, Doncaster, Yorkshire, you enter Maxima Cæsariensis.
XVI.
Legolium, Castreford, Yorkshire.
 XXI.
Eboracum municipium, formerly colonia, leg. vi. victrix, York.
XVI.
Isurium, Aldborough by Boroughbridge, Yorkshire.
XXIV.
Cataractonium, Latio jure donat. Cateric, Yorkshire.
X.
Ad Tisam amnem, Piersbridge, Durham county.
XII.
Vinovium, Binchester, Durham county.
XIX.
Epiacum, Chester in the street, Durham county.
IX.
Ad Murum, Newcastle, Northumberland.
XXV.
Ad Alaunam, flu. Alnwick, Northumberland.
XXX.
Ad Tuedam, flu. Berwick, Scotland.
LXX.
Ad Vallum, Falkirk, Scotland.

ITER V.
From the Vallum, Falkirk, to Prætuarium, Patrinton. Vallum,
Antonini, Falkirk, Scotland.
. . . .
Corium, on the Watling-street, Romanhow, Korstonlaw.
. . . .
Ad Tines, Rochester on the river Tyne in Redesdale.